Practocal Manual On Plant Tissue Culture

Practocal Manual
on
Plant Tissue Culture and its certification
Dr. Kailash Ch. Samal

Dr. Gyana Ranjan Rout
Department of Agricultural Biotechnology

College of Agriculture
Orissa University of Agriculture & Technology, Bhubaneswar
2018
Training Manual
on
Dr. Kailash Ch. Samal

Dr. Gyana Ranjan Rout
Department of Agricultural Biotechnology

College of Agriculture
Orissa University of Agriculture and Technology
Bhubaneswar
2018
Practical Manual on
2nd Edition
2018
Copyright 2018 Orissa University of Agriculture and

Technology, Bhubaneswar, Odisha
All rights reserved. No part of this publication may be reproduced,

stored in retrieval system or transmitted by any means electronic,
mechanical, photocopying, recording or otherwise, without prior
written permission of publisher
Printed at:
M.S. Infotech, Bhubaneswar
CONTENT
Chapter Topic Page No
Chapter 1 Plant Tissue Culture 1
Chapter 2 Tissue Culture Laboratory 8
Chapter 3 Molecular Techniques 31
Chapter-4 Techniques for virus testing and elimination 37
Chapter 5 Micropropagation for quality banana plantlets 46
Chapter 6 National Certification System for Tissue Culture Plants 58
Chapter 7 Banana- Tissue Culture – (BTC) - Standards 66
Chapter 8 Sugarcane- Tissue Culture – (STC) – Standards 70
Chapter 9 Bamboo- Tissue Culture – (BaTC) - Standards 74
Chapter 10: Potato–Tissue Culture-Raised Mini-tuber- (PTCMT) 78
Standards for Certification
Chapter 11: Lab Experiments 82-115
1 General Instructions for Using Tissue Culture and 82
Molecular Biology Laboratory
2 Instrumentation for a Tissue culture Laboratory 86
3 Study of laboratory equipments for Genetic fidelity study 89
4 In vitro meristem culture of sugarcane (Saccharum 93
officinarum)
5 In vitro root regeneration of sugar cane from 97
micropropagated shoots
6 Meristem culture of ornamental plants in MS media. 100
7 Suspension culture technique for production of 102
secondary metabolites
8 Synthetic seed formation from somatic embryos 104
9 Extraction of genomic DNA from leaf samples by CTAB 106
method.
10 Purification of genomic DNA 110
11 Study of quality checking of DNA by agarose Gel 112
electrophoresis
12 Fidelity study of banana plantlets using ISSR primers 115
Annexure 118-126
CHAPTER 1:
Plant Tissue Culture
Traditionally, plants are multiplied by means of seeds (sexual propagation)

or organs other than seeds (asexual or vegetative propagation). These organs are
usually stems, leaves or roots. Though multiplication by seeds is the cheapest
method, it suffers from certain disadvantages. Plants raised from seeds may not
repeat good performance of mother plants. Many plants take a long time to
produce seeds/fruits and many of them do not produce viable seeds or desired
quality of seeds. Plants propagated vegetatively do not suffer from these
disadvantages. However, vegetative propagation is rather a slow, time and space
consuming process. Besides, it is usually infected with latent diseases. Some
plants are also not amenable to vegetative method of propagation, for example,
coconut, papaya, oil palm, clove etc.
Therefore, scientists started a quest for an alternative method of plant
propagation which could overcome the disadvantages of both the methods
described above. After many trials and errors in the sixties, plant propagation by
tissue culture method, which could overcome disadvantages of propagation by
seeds or vegetative organs, was found commercially successful in the case of
horticultural and forest plants.
Plant tissue culture is a modern technique that describes the range of
procedures used to maintain and grow plant tissues and organs (stems, roots,
embryos) in aseptic (sterile) cultures. Plant tissue culture is widely used for the in
vitro vegetative propagation of plants in a process known as micropropagation. In
this process, a part of the plant (explants) is cultured aseptically under controlled
environment on agar-gelled medium containing all the nutrients, vitamins and
phyto-hormones to produce callus or shoots which in turn converted to whole plant
after initiation of roots. So, micropropagation, a proven means of producing millions
of identical plants by culturing plant tissues or organs under germ-free conditions.
In this case, a small piece of plant tissue (explant) is taken from the donor plant
and cultured on a nutrient medium in sterile containers. By altering the composition
of the medium and the environmental conditions (temperature, light regime, etc.)
Practical Manual on Plant Tissue Culture and its Certification: Dr. K.C. SAMAL & Dr. G.R. ROUT
Page 1
the development of this piece of tissue can be directed along different patterns and
finally the whole plant can be regenerated. The offspring all come from a single
plant and thus have identical genetic make-ups to each other and to the mother
plant. They are thus called clones.
1.1. Various stages of micropropagation
Stage 0 : Selection and preparation of the mother plant
: Sterilization of the plant tissue takes place
Stage I : Initiation of culture
: Explant placed into growth media
Stage II : Multiplication
: Explant transferred to shoot media; shoots can be constantly

divided
Stage III : Rooting
: Explant transferred to root media
Stage IV : Transfer to green house and net house
: Gel washed TC plants were transferred to green house in

semisterile medium for acclimatization and hardening
Stage V : Transfer to soil
: Explant returned to soil; hardened off
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1.2.. Flow chart of Plant Tissue culture techniques
Practical Manual on Plant Tissue Culture and its Certification

Certification: Dr. K.C. SAMAL & Dr. G.R. ROUT
Page 3
1.3. Major advantages of tissue-culture:
A large number of true to the type plants could be propagated within a
short time and space and that too throughout the year. For example, it may be
possible to propagate 2-4 lakhs of Tissue Cultured Plants (TCP) from a single bush
of rose against 10 to 15 plants by vegetative means. Also, it may take about 2-4
months to produce healthy planting materials by tissue culture means, whereas a
minimum of 6-8 months is required for most species by the latest method of
vegetative propagation.
 Large number disease free plantlets can be produced in short time and
space.
 All the plantlets produces are uniform in nature and carry all the characters
of mother plant (true–to-the –type).
 The production of plantlets through tissue culture can be achieved
throughout the year (i.e., season independent).
 Rare hybrids of forest plants can be produced by employing embryo rescue
techniques.
 Homozygous plants can be produced from hybrids through pollen or anther
or ovule culture followed by chromosome doubling.
 Tissue-culture could be a useful way of circumventing or eliminating
diseases.
 Tissue culture plants (TCPs) may have increased branching and flowering,
greater vigour and higher yield, mainly due to the possibility of elimination of
diseases.
 The method may succeed to propagate plants where seeds or vegetative
propagation is not possible or difficult or undesirable.
 The method saves space and energy. For example, 2500 m2 of heated
green house can be replaced by a climatised room of 10 m 2
 The flexibility of nurseries can be improved. As the capital investment on
mother plants is reduced to almost zero, it may be easier to adapt to
changing conditions. Additionally, a better programming of the production is
possible, because of the greater plant uniformity and the availability in the
mass at any time.
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 Tissue culture can be utilized for breeding new varieties, preservation of
germplasm and in vitro synthesis of metabolites.
1.4. Applications of plant tissue culture

 Micropropagation is widely used in agriculture, horticulture and forest
plants.
 A plant breeder may use tissue culture to screen cells rather than plants for
advantageous characters, e.g. herbicide resistance/tolerance.
 Large-scale growth of plant cells in liquid culture in bioreactors for
production of valuable compounds, like plant-derived secondary metabolites
and recombinant proteins
 To cross distantly related species by protoplast fusion and regeneration of
the novel hybrid.
 To cross-pollinate distantly related species and then tissue culture the
resulting embryo which would otherwise normally die (Embryo Rescue)?
 For production of doubled monoploid (dihaploid) plants from haploid
cultures to achieve homozygous lines more rapidly in breeding
programmes, usually by treatment with colchicines which causes doubling
of the chromosome number.
 Certain techniques such as meristem tip culture can be used to produce
clean plant material from virus infested stock, such as potatoes and many
species of soft fruit.
 Micropropagation using meristem and shoot culture to produce large
numbers of identical individuals.
 Production of identical sterile hybrid species can be obtained.
 Micropropagation can also be used to conserve rare or endangered plant
species.
1.5. Commercial prospects of Tissue culture in Agriculture

Propagation by tissue-culture offers good commercial prospect in
agriculture, horticulture and also forest plants, where value of the products is high.
The technique has reportedly been successful in more than 100 species of plants.
It has been estimated that more than 350 million TCPs are being produced
annually through tissue culture.
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In India there are at least ten commercial organisations, which have
developed technical competence for tissue culture. The present installed capacity
is about 50 million Tissue Culture Plants (TCPs) and the export is of the order of
Rs. 50 crores. The working group appointed by the Ministry of Commerce has
proposed an export target of Rs. 300 crore i.e. about 60 million TCPs over a five
year period as against the present production of 8-10 million TCPs. Tissue culture
method of propagation is highly labour intensive, 55-60% of the cost is on account
of labour. India's potential for export of tissue culture plants is rated very high
because of abundant and cheap labour.
The domestic market for TCPs, though nascent at present, is likely to
develop, since tissue culture method of propagation can multiply an elite plant very
rapidly. Recently however, several plant tissue culture laboratories and commercial
facilities have been set up, which are generating quite a large number of tissue
culture-raised commercial crops and forest trees. However, India lacks organized
testing of the quality of regenerants and freedom from viral contamination. The
most deleterious variants in tissue culture-raised plants are those that affect yields,
quality, and carry viral infections that are difficult to diagnose.
1.6. List of plants for which technologies have been perfected for large
scale propagation
Plant category Plants
Fruits : Banana, Grapes, Pineapple, Strawberry, Sapota
Cash crops : Sugarcane, Potato
Spices : Turmeric, Ginger, Vanilla, Cardamom
Medicinal plants : Aloevera, Geranium, Stevia, Patchouli, Neem
Ornamentals : Gerbera, Carnation, Anthurium, Lily, Syngonium
1.7. Highlights of the market survey on TCPs:

The major consumers of tissue culture plants (TCPs) are the State
Agriculture Department, Agriculture Export Zones (AEZs), sugar industry and
private farmers. The paper industry, medicinal plant industry and State Forest
Departments are using TCPs in a limited scale. The requirements of TCPs for the
above consumer segments are primarily met by the commercial tissue culture units
and to a small extent by the Micropropagation
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PLANT PRIORITY PLANTS
CATEGORY
Horticulture : Banana, Papaya, Strawberry, Grapes, Apple, Sapota,
Mandarin Orange, Passion Cherry, Walnut, Almond, Pecan
nut, Pineapple, Fig
Spices : Vanilla, Ginger, Turmeric, Pepper, large Cardamom
Medicinal and : Patchouli, Gloriosa, Senna, Aswagandha, Aloe, Nightshade
Aromatic Plants (S. khasianum), Phyllanthus, Dioscorea, Neem, Geranium,
Safed musali, Taxus, Digitalis
Ornamental plants : Orchids, Gerbera, Anthurium, Chrysanthemum, Rose,
Dendrobium
Forest plants : Eucalyptus, Subabul, Acacia, Casuarina, Bamboo
Page 7
CHAPTER-2
Tissue Culture Laboratory
2.1. Laboratory set up

Any standard laboratory, in which tissue culture techniques are performed,
regardless of the specific purpose, must contain a number of basic facilities. These
usually include:
 Washing and storage of glass wares

 Media preparation, sterilization and storage area
 Aseptic preparation and inoculation of plant materials
 Culture rooms for maintenance of cultures under controlled conditions
 An observation/data collection area
This can be achieved by having four separate rooms. Depending upon
the type of work to be conducted, the laboratory requirements vary considerably.
2.1.1. Washing room

The washing room/area must be in a corner of the laboratory. The washing
room should contain a work bench fitted with a large washing sink, water tap, some
lead -line to resist acids and alkalis, draining boards, racks and have access to
demineralized water as well as double distilled water. Hot air ovens to dry washed
lab wares, automated dishwashers, acid baths, pipette washers, driers and plastic
buckets/tubs to soak the lab ware before washing and storage cabinets should also
be available in the washing area.
2.1.2. Media room

Nutrient media preparation and their sterilization are carried out in media
room. Preparation of media requires following mentioned facilities
 Work bench
 A refrigerator and freezer for storing temperature sensitive chemicals and
stock solution
 Double distillation unit for constant supply of distilled water
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 pH meter
 Weighing balance
 Filter sterilization units for sterilizing heat labile substances
 An autoclave or domestic pressure cooker for sterilizing media
 Magnetic stirrer to dissolve chemicals
 A dust free cabinet or shelf for storing inorganic chemicals
 Hot plates/ stirrers, balances, water baths and media dispensing equipment
 An air and vacuum sources, Bunsen burners with gas sources
 A microwave oven or convection oven for boiling the media and glass wares
In preparing culture media, analytical grade chemicals should be used and
good weighing practice should be followed. The water used in preparing the media
must be of the utmost purity and highest quality. Tap water is unsuitable because it
may contain cations (ammonium, calcium, iron, magnesium, sodium, chlorides,
fluorides, phosphates etc), microorganisms (algae, fungi, bacteria), gases (oxygen,
carbon dioxide, nitrogen), and particulate matter (silt, oils, organic matter etc). The
room is also provided with a high temperature hot air oven for sterilizing bottles,
tubes and other glasswares and a shelf for storing nutrient media to be used for
initiating culture.
2.1.3. Media storage room

Aseptic cleaned room with cabinets to store autoclaved media.
2.1.4. Inoculation room

Under very clean and dry conditions, tissue culture techniques can be
successfully performed on an open laboratory bench. However, it is advisable that
a laminar air flow hood or sterile transfer room be utilized for making transfers. An
interior small room away from the flow of air current is an excellent location for
aseptic inoculation. This room is provided with a laminar air flow hood, which gives
a gentle flow of ultra filtered sterile (HEPA filter, 0.3 μm) air across the working
area in the hood. Air is forced into the unit through a dust filter then passed through
a HEPA filter. The air is then either directed downward (vertical flow unit) or
outward (horizontal flow of unit) over the working surface. This type of culture hood
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is sterilized by an ultra violet light and wiped down periodically with 95% ethyl
alcohol when in use.
2.1.5. Culture room

The bottles or tubes containing inoculated explants on the semi-solid
medium are kept in culture room under regulated environmental conditions i.e.
temperature, humidity and light. These environmental factors influence the growth
and differentiation process directly during culture or indirectly by affecting their
response in subsequent generations. To maintain the room temperature around
22+2oC generally air conditioners/heaters are used. Temperature sensor and
sequential timers for air conditioners is essential and the temperature should be
constant through out the entire culture period. The relative humidity of the room
should be of about 85-90%. Protoplast cultures, low density cell suspension
cultures and anther cultures are particularly more sensitive to environmental
culture condition. Fluorescent tubes are fitted in top of the all shelves of culture
rack (150 × 90 × 45 cm). Temperature sensor and light timer are fitted in the
culture room, to regulate temperature and photoperiod respectively as per the
requirement of culture condition. The culture room should have enough fluorescent
lighting from 3,000 lux to reach the 10,000 lux. Both light and temperature should
be programmable for a 24-hours period for incubating cultures generally of 16
hours light and 8 hours dark. Some laboratories have illuminated selves on the wall
along the sides of the room.
2.2. Basic requirements

Glass wares (conical flasks, volumetric flasks, measuring cylinders,
beakers, graduated pipettes, test tubes, culture bottles, glass rods, and petri
dishes); cottons, tissue papers; trolley to transport cultures, media, culture bottles
etc; low speed centrifuge to sediment protoplasts, cells etc; simple microscope,
Inverted microscope to observe tissues and cells, haemocytometer for counting
cells and protoplasts; Deionizer system for de ionized waters
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2.2.1. Plant tissue culture media
Nutritional requirements for optimal growth of tissues in vitro may
vary with different species of explants. In literature several information on different
plant tissue culture media is available like Nitsch and Nistch (1956), White (1963),
Murashige and Skoog, medium (MS) (1962), Gautheret (1942) etc. But MS
medium has proved effective for the growth of different tissues and is widely used
for different tissue culture work. The details of the MS medium are described
below. The components of MS Media can be divided in to six major groups such
as:
2.2.2. Inorganic macro nutrients

Inorganic chemical and organic chemicals have different structural
elements. Most inorganic chemicals have high melting points, few will burn, many
are soluble in water, most are insoluble in organic solvents, and reactions involving
inorganic chemicals tend to be very fast. The important nutrient required in macro-
molar concentrations include N, P, K, Ca, Mg and S. Nitogen (N) influences the
rate of plant growth. It is an essential element in the molecular make up of nucleic
acids, proteins, chlorophyll, amino acids, alkaloids, and some plant hormones.
Phosphorous (P) is an abundant in meristematic and other fast growing tissues as
part of DNA and ATP (Adenosine tri phosphate) molecules. Potassium (K) is
necessary for normal cell division and promotes meristematic growth. It plays a
role in many reactions within plants, although it is not an actual component of plant
protoplasm, fats or carbohydrates. Calcium (Ca), in the form of calcium pectate, is
an integral component of plant cell walls. Dead shoot or root tips can be a sign of
lack of calcium. Magnesium (Mg) is the central element in chlorophyll molecules. It
is also important as an enzyme activator. Magnesium deficiency causes older
leaves to become chlorotic. Sulpher (S) is present in some proteins. It promotes
root development and deep green foliage. These elements are made available in
MS Medium by its ingredients, Potassium Nitrate(KNO 3), Amonnium
nitrate(NH4NO3), Calcium chloride (CaCl2.2H2O), Potassium dihydrogen phosphate
(KH2PO4) and Magnesium sulphate (MgSO4.7H2O). The concentrations of these
nutrients in MS medium are as follows
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Macronutrients mg/l
KNO3 1900
NH4NO3 1650
KH2PO4 170
MgSO4.7H2O 370
CaCl2.2H2O 440
2.2.3. Inorganic Minor nutrients

In addition to the major elements that plants require, a number of other
elements are essential to good growth but are needed only in extremely small
quantities. The nutrients required in micro molar concentrations include Fe, Mn, Zn,
B, Cu, Mo, Cl. Iron (Fe) is involved in chlorophyll synthesis. Manganese (Mn)
deficiency is characterized by mottled yellowing of leaves. It is an essential
element in chloroplast membrane. Zinc (Zn) is an enzyme activator involved in
chlorophyll formation, as well as in the production of the auxin indole-3-acetic acid
(IAA). Boron (B) is an important trace element presumed to play a role in the
movement of sugar, water, and, hormones. Copper (Cu) deficiency results in
stunted growth, malformations, twisted and blotched leaves. Molybdenum (Mo) is
required for normal growth and protein synthesis. Chlorine (Cl) helps to stimulate
photosynthesis and seems too necessary for growth. The requirement of
microelements is met by addition of following salts in MS medium.
Micro Nutrients mg/l
MnSO4.7H2O 22.3
ZnSO4 8.6
H3BO3 6.2
KI 0.83
Na2MoO4.2H2O 0.25
CuSO4.5H2O 0.025
CoCl2.6H2O 0.025
2.2.3. Iron Sources

Iron is essential for cell division and usually supplied as the chelate (Na-
FeEDTA). This ensures availability of iron at pH values up to 8.0 and through out
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the growth of culture. Iron depletion occurs rapidly in the absence of a chelating
agent. In MS medium Na-FeEDTA is prepared using following ingredients:
Ingredients mg/l
FeSO4.7H2O 27.8
Na2EDTA.2H2O 37.3
2.2.4. Organic supplement (Vitamins)

Vitamins have catalytic functions in enzyme systems and are required only
in trace amounts. Some consider that Thiamine (vitaminB1) may be the only
essential vitamin for nearly all plant tissue cultures, where as Nicotinic acid (niacin)
and Pyridoxine (Vitamin B6) may also stimulate growth. The organic supplements
of MS medium are as follows
Organic Constituents mg/l
Myo-Inisitol 100.0
Thiamine-HCl 0.1
Nicotinic acid 0.5
Pyridoxine-HCl 0.5
Glycine 2.0
2.2.4. Carbon Sources

All media require the presence of carbon as energy source. The most
commonly used carbon source is sucrose. Glucose and fructose are also known to
support good growth of some tissues. In MS medium, generally 30 g sucrose per
liter is added as carbon sources.
2.2.5. Plant growth regulators

Growth regulators, or hormones are not nutrients, but they influence
growth and development. They are generally produced naturally in plants during
their growth. But under in vitro condition, growing explants does not able to
manufacture sufficient quantities of growth regulators. So they must be added
selectively to culture media. These are the auxins, cytokinins, gibberllins, abscisic
acid, ethylene etc. Auxins are phytohormones that influence cell elongation,
swelling of tissues, root initiation, and adventitious bud formation. Auxins are
Page 13
commonly used in tissue culture media either combined with cytokinins during the
multiplication stage or without cytokinines for the rooting stage. Cytokinins, formally
called kinins are growth regulators that are required in tissue culture media for cell
division, shoot multiplication and auxiliary bud proliferation. They usually omitted
from media for the rooting stage.
2.3.1. Stock solution and media preparation

The most efficient way of preparing plant cell culture media is first to make
up stock solutions of macro nutrients, micronutrients, vitamins, iron source and
individual plant growth regulators. Several tissue culture media are available
commercially in the form of dry powders containing all components except growth
regulators, Sucrose and agar. However, they are expensive and are of limited use
for studies involving manipulations of media components. A more convenient
method of preparing concentrated stock solutions and mixing appropriate
quantities of stock solutions to get desired volume of the medium.
2.3.2. Nutrient stock solutions

To prepare MS basal medium four different stocks are used. These are:
2.3.3. MS macro (MS-I, 20X)

The stock contains all major inorganic nutrients. MS-1 stock for 20 liters of
medium is prepared at a time as indicated below:
KNO3 - 38.0g
NH4NO3 - 33.0g
CaCl2.2H2O 8.82 g
MgSO4 - 7.4g
KH2PO4 - 3.4g
↓ ↓
Dissolve in about 1000 ml of double Dissolve in about 500 ml of double
distilled Water. distilled Water.
Mix the solutions and make volume to 2000 ml with DD water and store at
4oC. Use 100 ml stock for preparing 1 liter MS medium.
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2.3.4. MS micro (MS-II, 100X)
All micro nutrients except FeSO4.7H2O and Na2EDTA 2H2O are dissolved
together to prepare MS-II stock. MS-II stock for 100 liters of medium is prepared
as under.
MnSO4.4H2O 2.23g
ZnSO4.7H2O 860mg Use 10 ml
Dissolve all Stock for
H3BO3 620mg chemicals in DD preparing
KI 83mg → water and make → 1liter
Na2MoO4.2H2O 25mg* Volume to 1000ml. MS medium
Store at 40 C
CuSO4.5H2O 2.5mg*
CoCl2.6H2O 2.5mg*
*The small quantities of chemicals are difficult to weigh accurately; it is
preferable to prepare 100ml stocks containing 1mg of the chemical per ml and use
appropriate volume of the stock.
2.3.5. MS iron (MS-III, 20X)

The chelate solution Fe/EDTA can be easily prepared in the laboratory.
The MS III 20 liter stack medium is prepared as follows:
Na2EDTA.2H2O = 746 mg FeSO4.7H2O = 556mg
↓ ↓
Dissolve in about 80 ml of double Dissolve in about 80 ml of double
distilled Water. distilled Water.
Add FeSO4 solution to Na2EDTA solution with vigorous stirring. Make
volume to 200 ml with DD water. Store the stock in coloured bottle/ aluminium foil
rapped bottle at 4oC. For preparing 1 liter MS medium use 10 ml of this stock.
2.3.6. MS Vitamins (MS- IV, 100X)

Generally this stock is prepared by dissolving the below mentioned
quantities of vitamins in DD Water for 100 liters of MS Medium.
Myo-inositol 10.0g Dissolve all Use 10 ml

Thiamine 10.0mg chemicals in Stock for
DD water and → preparing 1liter
Nicotinic acid 50.0mg →
make Volume to MS medium,
Pyridoxine HCl 50.0mg 1000ml. Store at store the stock
Glycine 200.0mg 4 oC in deep freezer
Page 15
2.3.7. Growth regulators stocks
One or more growth substances are added to the basal medium to support
good growth of tissues. These growth regulators include auxins, cytokinins and
gibberllins. The plant growth regulators commonly used in tissue culture media are
summarized in Table 1.
Table 1: Plant growth regulators commonly used in tissue culture media
Conc. of Storage temp.
Name of the growth Stock
Class Abbreviation Mol. Wt. Solvent* Sterilization
regulator solution Chemical Stock
(mg/ml) solution
P-chlorophenoxy 50% Room
CPA 186.6 0.1-0.5 Autoclave 0-5 oC
acetic acid. ethanol Temp
2,4-Dichlorophenoxy 50% Room
2,4-D 221.0 0.1-0.5 Autoclave 0-5 oC
acetic acid ethanol Temp
Auxins
Deep Deep
Indole 3-acetic acid IAA 175.2 1N NaOH 0.1-0.5 Autoclave
Freeze Freeze
Deep
3-indolebutyric acid IBA 203.2 1N NaOH 0.1-0.5 Autoclave 0-50 C
Freeze
x-naphthalene acetic Room
NAA 186.2 1N NaOH 0.1-0.5 Autoclave 0-5 oC
acid Temp
6-Benzyl amino
Room
purine(Benzyl BAP (BA) 225.2 1N NaOH 0.1-0.5 Autoclave 0-5oC
Temp
adenine)
N-isopentenylamino- Deep Deep
Cytokinins
2iP 203.3 1N NaOH 0.1-0.5 Autoclave

purine Freeze Freeze
6-Furfuryl amino- Deep Deep

K 215.2 1N NaOH 0.1-0.5 Autoclave
purine(kinetin) Freeze Freeze
Filter Deep Deep

Zeatin Zea 219.2 1N NaOH 0.1-0.5
sterilize Freeze Freeze
Room
GA Gibberellic acid GA3 346.4 50% ethanol 0.1-0.5 Autoclave 0-5 oC
Temp
2.3.8. Auxins
In tissue culture commonly used auxins are IBA (Indole-3-Butyric acid),
IAA (indole acetic acid) NAA (naphthalene acetic acid) and 2,4-D .The compound
most frequently used and high effective is 2,4-dichlorophenoxyacetic acid(2,4-D).
The above auxins can be made in the same way as described here for NAA.
Page 16
2.3.9. NAA Stock (0.1mg/ ml)
Using a dropper, slowly add Finally volume make up
While stirring with a spatula, to
IAA
→ Several drops of 1M NAOH or → 250 ml by using dd
25g
KOH until the NAA crystal are Water. And stored In a
dissolved refrigerator
2.3.10. Cytokinins
Cytokinins are the derivatives of adenine. These compounds are also used
for overcoming apical dominance. Most frequently used cytokinins in plat tissue
culture are BAP (Benzyl amino purine), Kinetin (Furfuryl amino purine), Zeatin etc.
2.3.11. 6-benzyl-aminopurine (BA) (0.1mg/ ml)
Finally volume make up

Add 1M HCL Solution one to
BA Drop until the BA is dissolved.
→ → 250ml by DD Water.
25mg Apply a little heat to help
dissolve the crystals. And stored In a
refrigerator
2.3.12. Gibberlins
Although many gibberlins are known, GA3 is commonly used in tissue
culture media. It induces elongation of internodes and the growth of meristams or
buds in vitro.
2.3.13. Preparation of media

Preparation of tissue culture medium from individual stock solution is very
simple and involves following steps. The method is exemplified for prepairing 2 litre
MS Medium
2.3.14. Stock solution mixing

The various nutrient stocks are mixed in about 500ml DD water one
after another for preparing one liter basal MS Medium.
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The stocks to be mixed are:
MS-I 100ml
MS-II 10ml
MS-III 10ml
MS-IV 10ml
Required quantities of sucrose are added to this and dissolved using
magnetic stirrer. Necessary amount of growth regulators are added according to
the purpose of the medium and finally volume made to one liter using DD water.
Thermo labile growth regulators like zeatins are added after filter sterilization.
2.3.15. pH Adjustment
pH of the above medium is adjusted to 5.8 by using 0.1N NaOH and 0.1N
HCl before autoclaving. The pH determines many important aspects of the
structure and activity of biological macromolecules. The pH higher than 7 and
lower than 4.5 generally stops growth and development.
2.3.16. Pouring and Autoclaving

Agar is dissolved in the pH adjusted medium for preparation of semi-solid
media. In one liter medium usually 6-8g agar is dissolved by heating until near
boiling with intermittent stirring and pour into suitable containers like tubes, flasks,
bottles etc. For preparing liquid media pH adjusted media is poured in suitable
containers without dissolving agar in it. If petridishes are to be used in the
experiment, the Petri dishes and media are autoclaved separately then the
autoclaved media is poured in sterile Petridishes under laminar air flow hood. Plant
tissue culture media are usually autoclaved at 121oC (15 Lb/inch2 or 1.05Kg/cm2).
Minimum time required for steam sterilization of media as suggested by Biondi and
Thorpe (1981) is given in Table 2. After autoclaving store the sterilized medium at
25 to 30oC for further uses.
Page 18
Table 2: Minimum time necessary for steam sterilization of media
Volume of medium(ml) Time (minutes)
20-50 15
75 20
250-500 25
1000 30
1500 35
2000 40
2.4. Initiation of Tissue Culture

Explants are a piece of plant from which a culture is started. Explants
range in size from a microscopic 10th of a millimeter to stem pieces of several
centimeters in length. Explants can be meristems, shoot tips, macerated stem
pieces, nodes, buds, flowers, peduncle (flower stalk) pieces, anthers, petals, piece
of leaf or petiole, seeds, nucellus (the central part of an ovule) tissue, embryos,
seedlings, hypocotyls, bulblets, bulb scales, cormels, radicles, stolons (runners),
rhizome tips, root pieces, single cells or protoplasts. Initiation of cultures involves
selection, preparation, sterilization and inoculation of explants.
2.4.1. Selection of Explants

The choice of explants depends mainly on the aim of research.
Types of culture Explants used
Callus culture Leaves, anthers, root segments, petioles, tuber discs
Cell culture callus derived from above explants
Meristems tip cultrure Apical /auxiliary buds from stem segments or Sprouts
Protoplast culture callus/cell cultures, leaves
Micropropagation Nodal cutting from meristems, sprouts, apical buds
Conservation of Nodal region from infection free in vitro plantlets
germplasm
Regeneration Leaf, steam and tuber fragments, protoplasts callus
Embryo culture Mature or immature seeds
Anther culture Anther or pollen grains
Page 19
Only disease free plants should be tissue cultured. Before taking explants,
stock plants should be moved to a clean green house, screen house, or other
shelter away from dust and disease. They can be observed and given special care
usually about 2 weeks to 6 months in controlled conditions the plant will become as
healthy as possible, and they will not be stressed. Wash the plants with clean
water, allow the foliage to dry, and thereafter feed and water them only at the base.
The new shoots that appear will provide relatively clean explants and, in addition,
may provide some increased juvenility.
 The smaller the explants, the less contamination to remove; but the larger
the explants, the more tissue there is to help establish it in culture
 Use new shoots whenever possible because they are cleaner than old
wood.
 Plant under cover or dormant branches usually cleaner than field plants
 Explants from younger plants respond more quickly than older plants
 Explants may do better if taken in the morning
 Cut explants with cutters that have been sterilized by Deepings in 1/10
bleach
 Upon cutting explants material from the source plant, place the explants in a
plastic bag containing a moist paper towel until you are ready to work on it
to the laboratory
 Keep explants in deep freezer until you are ready to process it, but process
explants as soon as possible for best results
 5 mm size leaf petiole sections are recommended for explants, cutting to
5mm pieces should not be done until final processing in the hood.
2.4.2. Explants cleaning and treatment

The explants are separated from the source plant, they must be submitted
to cleaning treatments. Surface of the explants carry a wide range of microbial
contaminants. Therefore the explants should be surface sterilized before
inoculating. Visual symptoms of fungal and bacterial infections are not used.
various sterilizing agents/chemicals are used to sterilized explants the sterilizing
agent are toxic to plant tissue, therefore proper sterilizing agent concentration and
Page 20
treatment duration should be standardized to minimize explants injury. Some of the
commonly used sterilizing agents and treatment duration are given in Table 3
Table 3: Types of surface sterilants used for aseptic culture

Sterilizing agent Concentration Duration Effectiveness
(min.)
Calcium hypochlorite 9-10% 5-30 Very good
Sodium hypochlorite 2% 5-30 Very good
Hydrogen Peroxide 10-12% 5-15 Good
Bromine water 1-2% 2-10 Very good
Silver nitrate 1% 5-30 Good
Mercuric chloride 0.1-1% 2-10 Satisfactory
Antibiotics 4-50mg/l 30-60 Fairly good
A rapid rinse (about 40 second) of explants in 75% ethanol before
treatment with few drops of triton-x or tween-20 to the sterilizing agents enhances
the efficiency of sterilization. After sterilizing agent treatment the explants are
thoroughly washed 4-5 times with sterile double distilled water to remove the toxic
sterilants adhering to the explants. The sterilizing explants are now ready for
inoculation on nutrient media.
2.4.3. Inoculation of explants

Inoculation is carried out under aseptic conditions of laminar air flow hood.
Before explants inoculation, all instruments like forceps, scalpels, scissors, needles
etc. are sterilized by deeping them in 95% alcohol and flaming and cooling it .The
sterilized explants are transferred to a pre sterilized petridishes. The suitable size
of explants are prepared by dissecting for inoculation. Dissecting microscope is
used for dissecting out fine explants like meristems tips. Before dissecting the
working space of the microscope is sterilized by washing it with 95% ethanol or UV
light expose. Lids of culture vessels /cotton plugs are removed from the
bottle/conical flask/test tubes and the sterilized explants are transferred on to the
medium. The neck of the vessel (glass test tubes/conical flasks/bottles) is flamed
and lid/plug replaced quickly. The cultures are now ready for incubation.
Page 21
As we have emphasized, cleanliness is of primary importance in the
transfer room, especially in the hood, where cultures are momentarily outside of
their sterile protective containers-a time they have the greatest danger of becoming
contaminated. It is not easy to imagine and deal with the unseen. One has to
assume that clothing, skin, counter tops, instruments, and the air are teeming with
mold spores, bacteria, and a host of other invisible enemies ready to invade clean
cultures.
2.4.4. Practices to be strictly observed in the laboratory

 No eating, drinking, smoking, or storing food in the laboratory
 Wear a protective apron when working with cultures. Long full sleeves
should be avoided because contamination will be possible.
 Wear sterilized disposable medical gloves.
 Cover long hair and tie-back. Loose hair is a source of contamination for
cultures.
 Sit straight and keep your hand out of the transfer hood. Work at arm ’s
length, as far back in the hood as is practical. avoid wide sweeping arm
movements over the work area
 Set necessary equipment only in the transfer hood
 Sterilize in an autoclave or pressure cooker any bottles and Petri dishes
containing contaminated cultures before emptying.
 Disinfect work surfaces before and after working in the hood
 Keep transfer room door closed to avoid unnecessary drafts
2.4.5. Sterilization technique for explants
Protocol
 Turn on the transfer hood blower 10 min before transferring
 Rinse gloved hands in 10% ‘Teepol’ detergent
 Wipe inside the hood with 10% bleach, 70% alcohol, or Lysol
 Rise gloved hand in 10% bleach
 Immerse scalpel and forceps in 10% bleach for at least one minute and
rinse in 1% bleach.
Page 22
 Rinse the treated explants in 1% bleach using forceps, then sterile distilled
water rinses. Leave the explants in the sterile water rinse
 Using forceps and knife lay a paper towel on the laminar hood and place
explants from the sterile water on to the paper towel
 Using forceps and knife trim the explants in appropriate size. For shoot tips
you can use 1mm to 3 cm.
 With the left hand, grasp a test tube containing medium for the explants
hold the test tube near its base. to prevent any contamination from falling in
to the test tube, hold it at about 45o angle and facing right parallel with the
front of the hood so that it is facing neither the back filter nor you.
 Take forceps in the right hand. While still holding the test tube and the
forceps, grasp the test tube cap with the right-hand little finger, push and
twist the cap slightly.
 Still holding the forceps, test tubes, and cap, use the forceps to remove
explants from the sterile paper towel and place the explants firmly on the
agar in the test tube
 Still holding the forceps replace the cap on the test tube and place the tube
in the test tube rack
 Return forceps to 10% bleach
2.4.6. Sterilization techniques for sub-culture or transfer
Protocol
 Place the test tubes containing cultures ready for transfer on a counter out
side of the transfer hood
 Fresh sterile test tubes containing agar medium ready to receive the
transfers
 With the left hand, grasp a test tube containing the culture to be transferred.
Check the label to confirm that the test tube contains the correct culture.
 Using sterile forceps remove the culture from the test tube and place it on
the paper towel in the transfer hood
 Pass the forceps to the left hand and pick up the sterile knife with the right
hand cut, trim (trim away any brown and dead material) and divide the
culture as necessary.
Page 23
 Return knife to 10% bleach.
 Using forceps, place the culture in a test tube with the appropriate medium
some shoot transfers should be laid on the surface of the agar to induce
bud break if there are lateral nodes. Most shoot tips should be inserted in to
the medium with their bases just deep about 1 cm in the agar to allow the
shoot to be held upright. Transfers that are going in to a rooting medium
should be held upright.
 With forceps still in the right hand, replace the cap on the tube and place the
tube in the rack, ready for labeling.
 After doing 6 test tubes, place the towel in the disposable material on it in to
the dust bin, and get a fresh sterile paper towel in the hood.
 When you have finished transferring all the tubes of one variety, label each
test tube with date code, culture name and your name and transfer the
culture to the shelves in the culture growing room.
2.4.7. The growing room

The activity in the culture growing room depends on the work in the media
preparation and transfer rooms and the success of culture growth determines the
success of the entire work-it is the reason for precise media making and
painstaking transferring, and is the basis by which viable plants will sell. Good
tissue culturists spend a part of each day monitoring the cultures in the growing
room. Attention should be need to the lighting and temperature requirements of the
particular tissue culture plants. Culture seems to have a wide tolerance of light
intensity. Plant in culture have low range of photosynthesis probable because due
to sucrose in media.
2.5. Dealing with problems

Contaminated cultures should be removed as soon as they are discovered.
It is easy to tell when cultures are over run by bacteria, yeast, or molds growing on
the surface of agar. Other contaminants that grow as a cloud within the medium,
often surrounding the submerged base of the culture, are more difficult to see,
especially if the agar is not perfectly clear. The best way to detect the contaminant
is to look through the medium while holding the test tube up to a light. Bacteria are
easily observed when clear gel is used because many bacteria grow below the gel
Page 24
surface. The best way of action against contamination is to sterilize and discard all
contaminated cultures immediately. Reexamine the sterile procedure and check for
possible sources of contamination. All steps should be taken to prevent the spread
of existing contaminant and the introduction of new ones.
Although by no means a cure-all, antibiotics are effective against at least
some woody plant bacteria (Young et al.1984) especially when used both as a
presoak and liquid media. gentamicin sulfate, streptomycin sulfate, and penicillin-
streptomycin solution have also been credited with some success. A special
method of testing for contaminants, called indexing, is employed by some tissue
culturists.
2.5.1. Media problems

A careful attention should need for bleeding, the phenolic exudates from
the cultures that change the media colors in to inky purplish black. This condition
due to result of dull knives, old cultures, or too liquid medium. Bleeding has an
adverse effect on the culture, turning the base black and retarding its growth and
multiplication. It is need to transfer such culture as soon as the symptom is
observed.
Other culture conditions to look for are changes in color and growth
pattern. when leaves turn to yellow or brown, stem turn red, unwanted root or
callus are produced, it is usually an indication that it is time to vary the formula or
environment. If culture produces roots prematurely, the plantlets tend to mature
and will cease to multiply. Transferring the plantlet in to a media containing higher
cytokinin for brings back multiplication. Hormones create a dramatic influence on
culture growth. A methodical way to establish the optimum cytokinin/auxin ratio is
to draw a grid and plot an on the two axis in increasing increments (Table 4).
Page 25
Table 4: Auxin and cytokinin influence on culture growth
Cytokinin (mg/l)
Auxin 0 0.5 1 3 5 10
(mg/l)
0.0 0/0 0.5/0 1/0 3/0 5/0 10/0

0.5 0/0.5 0.5/0.5 1/0.5 3/0.5 5/0.5 10/0.5
1.0 0/1 0.5/1 1/1 3/1 5/1 10/1
3.0 0/3 0.5/3 1/3 3/3 5/3 10/3
5.0 0/5 0.5/5 1/5 3/5 5/5 10/5
10.0 0/10 0.5/10 1/10 3/10 5/10 10/10
For shoot culture in the multiplication stage (Stage-II), rule out ratios
where the auxin content is higher then the cytokinin content. A preliminary test
might include the six ratios underlined in Table 4. Culture in rooting media often
benefit from the addition of charcoal. The darkness of charcoal in rooting area may
also be a beneficial factor. Charcoal has also been used remedially for some
problems in the multiplication stage. There are of course many other problems in
addition to determining the correct medium for a given cultivar.
2.6. Symptoms, causes and solutions

There are many problems for which no specific solutions are known. The
following list of problems and possible solutions is mentioned in Table 5. As
problems arise you will want to do the following:
 Examine laboratory records to learn when a problem started.

 Check all instruments to be sure they are functioning properly, particularly
the pH meter, sterilizer and water purifier.
 Try variations of light, medium and temperature.
 Study the green house and field habits and requirements of the plant in
question to see if these factors yield any clues for treatment in culture
 Consult outside sources of help, including: Tissue culture supply companies
Other tissue culture laboratories Universities, other related departments.
Page 26
Table 5. Common tissue culture problems and possible solutions
Symptoms Possible cause Possible solution
Disinfectants too high Use weaker disinfectant
Medium too strong Use weaker medium (1/2 or ¼
Explants dies
strength
Timing Obtain explants at different times
Contamination Discard culture, maintain sterile
technique
Bleeding Transfer immediately
Culture blackens
and dies Water problem Check water purity
Too hot Reduce temperature
Wrong formula Change medium
Dormant Chill for a month
Start explants at different time
Needs more time Transfer explants
patience
Explants live but Wrong formula Change medium
no growth Decrease salt and hormones
Add charcoal
Add gibberellic acid
Medium too strong Use weaker medium
Too hot and cold Change temperature
Too hot or cold Change temperature
Grow too slow Wrong medium Change medium and keep
patience
Shoots too long Too little cytokinine Increase air or cytokinine
and spindly Too hot or cold Run cytokinine/auxin grid
Poor shoot Too little cytokinin Increase air or cytokinine
multiplication Run cytokinine/auxin grid
Too much cytokinine Decrease cytokinin
Shoots too short
Run cytokinine/auxin grid
Page 27
Less cytokinin Increase cytokinin
More cold Run cytokinine/auxin grid
No multiplication Needs chilling Increase temperature
Requires dormancy Cold store 5-8 weeks
Cold store 4-8 weeks
High cytokinin Decrease cytokinin
Fat stems
Run cytokinin/auxin grid
Hormone wrong Decrease cytokinin
Unwanted callus Run cytokinin/auxin grid
Omit auxin
Contamination Check for contamination
Chlorotic leaves Wrong formula Change medium
Too hot Reduce temperature
Osmosis upset Reduce temperature
Much cytokinin Increase air
Vitrification Lack of lignin in cell wall Increase agar
Culture too old Transfer more often
Wrong agar Change agar
Premature Wrong hormone balance Increase cytokinin
rooting Run cytokinin/auxin grid
High sugar Decrease sugar
Old culture Transfer more often
Red stems
Stress Change light and temperature
Little amount of nitrate Increase nitrate
No roots Grow root in culture
Too dry too heat Adjust humidity and soil moisture
Stage IV, plant Wrong ph, soil mix, Too Research and experiment
dies rapid hardening moister In stage III medium, decrease
pathogens sugar, open bottle 1-3 weeks
Test for pathogens
Medium Change medium
Aberration
Lack of air Provide sterile air
Senescence Increase or change hormones
Poor
pathogens Add charcoal
multiplication
Test for pathogens
Page 28
2.7. Hardening
Most growers are well acquainted with the hardening or acclimatization,
requirement in the green house for seedlings and cuttings. It is much easier to
grow seedlings than it is to establish tissue culture plantlets in green house
conditions. If care is not taken during this critical stage, high losses can result. An
abrupt change to the lower humidity and higher light of the green house can be
fatal to plantlets in a very short time.
The quality of stage IV plantlets depends on the quality of the plantlets that
come from the growing room. Often times stage III is omitted and plantlets are
rooted in soil directly from stage II. Sometimes the roots that develop in stage III
are not functional in soil, in which case the plantlet may go to soil from stage II.
Acclimatization can be started while plantlets are still in vitro, or it can wait until
stage IV, when plantlets are moved from container to soil. A major problem faced
in acclimatization is water loss.
There are several ways of preparing for acclimatization; if plantlets are to
be rooted in vitro in stage III .small bags of desiccant (silica gel) can be hung in
containers to lower the humidity. The cultures may dry quickly, so watch this work
carefully. covers of the container can be placed more loosely and allow more water
vapor to escape, but if left for more than few days contamination may be possible,
depending on the room. Depending on the room humidity, it is best to remove the
lids gradually. Another effective way to use lids with filters, which allow more
desirable air exchange and can be applied much earlier than removing the lids.
Common procedure for transferring plantlets from the culture growing room
to the green house is to wash off the agar from the roots, plant the plantlets in
artificial soil in undivided planter trays, and place them in high humidity in tents on
benches in shaded greenhouse. Over a 2-4 week period, the sides of the tank
should be gradually opened and the amount of mist gradually reduced to lower the
humidity, thus allowing the existing leaves to adjust and assisting new leaves to
grow. These encloses are equipped with mist, cooling pads, exhaust fans and
lighting with appropriate timing. The entire green house might be equipped in lieu
of a tent there is very difficult to control the humidity and moisture in larger green
house.
Page 29
A fog system is ideal way of maintaining high humidity. Fog is expensive
and difficult to maintain when exhaust fans are working to reduce heat.
Greenhouse shade can be maintained at 50%.if desired the photoperiod may be
extended with artificial light. A growing room equipped with fluorescent lights over
shelves, is a good option for establishing tissue culture plantlets from stage II or III
in to the soil. Humidity can be maintained by planting in 11 × 22 in (28 × 55cm)
seedling trays with plastic covers called Humidomes. A transparent smaller
container 4 × 4 in (10 × 10 cm) plastic Barry cup can hold up to 25 plantlets. A
second cup inverted in to the top and taped makes a miniature green house.
Drainage holes in the inverted tope’s cup should allow room for a wash-bottle stem
to be inserted for watering. They can also be watered from by flooding in a water
tight bench. The smaller container will help confine any disease problems to fewer
plants. The seedling trays ideally should house about 200 and the cups about 20
plantlets. An open bottom tray inverted over a tray of plantlets will serve as a
holder for this cover while allowing light and water through and it prevents droplets
from falling on the plants, holds humidity and moisture and modifies temperature.
Plantlets are moved from stage II or stage III to soil mixes; it is desirable to wash
off the agar as much as possible because yeast, moulds, bacteria and insects
thrive on the nutritious agar. Another approach is to transplant shoots to pots of
soils mix that are then placed in jars and covered as they had been in culture.
Page 30
CHAPTER-3:
Molecular Techniques
3.1. Protocol for DNA isolation

The young emerging healthy leaves were collected and immediately
brought to the laboratory. The leaves were washed with distilled sterile water and
cleaned with moist tissue paper. The leaf sample (2-3 g) was cut into small pieces
and placed in a pre-cooled mortar, frozen by adding liquid nitrogen and crushed
vigorously with pestle to a fine powder. The ground powder was transferred into a
clean sterile 50 ml centrifuge tube and 10 ml of pre-warmed (60 oC) extraction
buffer (details given below) was added to it. The content was shaken vigorously by
inversion to form slurry. The tubes were incubated at 60oC in circulating water bath
for 1 h with intermittent shaking to form an emulsion. After that equal volume of
chloroform: isoamyl alcohol (24:1) was added and mixed by gentle swirling for 15
to 20 min. After complete emulsion formation, centrifugation was done at 10,000
rpm for 10 min at 25oC.
The aqueous phase was transferred to a fresh centrifuge tube and then
equal volume of chilled isopropanol was added. The content was mixed by gentle
inversion. The tubes were kept for overnight at 4oC. The DNA was spooled out
carefully and kept in 1.5 ml eppendorf tubes, which were then spun at 10,000 rpm
for 10 min. The aqueous part was decanted. The pellet was washed with 70%
ethanol. The tubes were spun @ 10,000 rpm for 10 min at 25 oC. The aqueous part
was decanted and the pellet dried free of ethanol. The DNA pellet was dissolved in
100 μl of T10E1 (10:1) buffer (pH 8.0) and kept overnight for complete dissolution.
All the dissolved samples were treated with RNase A. One microlitre
solution of RNase enzyme (10 µg/ml) per 100 µl was added to the DNA solution
and mixed gently. The content was incubated at 37ºC for 1 h for the removal of
RNA. Equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added
and mixed properly for 5 min. Sample tubes were centrifuged at 10,000 rpm for five
min and the supernatant was collected in fresh tubes. Equal volume of chloroform:
isoamyl alcohol was added, mixed well and centrifuged for 10 min. The aqueous
layer was removed and the process was repeated to produce a creamy solution.
Page 31
Then one-tenth volume of 3M sodium acetate and 2.5 volume of absolute chilled
ethanol were added. The content was mixed gently to precipitate DNA and there
after kept overnight at -20oC.
The solution was then centrifuged at 8000 rpm for 5 min and the
supernatant was decanted off. Extra salts were removed by two washing with 70%
ethanol. DNA was dried under vacuum and dissolved in TE (10:1) buffer at room
temperature and stored frozen at - 20oC.
3.2. Quantification of isolated DNA and quality checking

The concentration of DNA was estimated by the measurement of the UV
irradiation absorbed by nucleic acid bases. First the spectrophotometer was
calibrated using 2000 l of TE in a quartz cuvette at 260 nm and 280 nm. Then five
l of DNA sample was added to 1995 l of TE, mixed well and absorbance (OD)
was taken at 260 nm and 280 nm. The concentration of the DNA in the sample was
estimated as follows
Concentration of DNA ( g/ml ) = OD at 260 × Dilution factor × 50

The ratio between readings at 260 nm and 280 nm (OD260/ OD280) provided
an estimate for the purity of nucleic acid. Any sample showing the ratio below 1.8
or above 2.0 was further subjected to purification.
Further in order to know intactness of genomic DNA, presence of proteins
and/or RNA contaminants, an aliquot (2 µl) of each sample was subjected to
agarose gel (0.8 % w/v) electrophoresis for about 2 h along with 500 ng of
molecular weight marker ( Lambda / EcoRI digest). The gel was stained with
ethidium bromide (0.5 g/ml), viewed under UV Transilluminator and photographed
immediately for further interpretation using a Gel-Doc system (Bio-Rad,USA). By
comparing the fluorescent intensity of the bands with the standard, DNA
concentration was also estimated following the method described by Sambrork et
al. (1989). Part of stock DNA samples were diluted with appropriate amount of TE
buffer to yield a working concentration of 10 ng/µl and stored at 4 oC.
Page 32
3.3. Polymerase Chain Reaction (PCR)
The PCR mixture consisted of Taq DNA polymerase, PCR buffer, dNTPs,
MgCl2, oligonucleotide primers and genomic DNA. Optimization of concentration of
PCR components was carried out for MgCl2, Taq DNA polymerase, and genomic
DNA concentration. To determine optimal amplification reaction conditions, a
factorial experiment was carried out at three concentrations of MgCl2 (2.0 mM, 2.5
mM and 3.0 mM), three concentrations of Taq DNA polymerase (0.5 U, 1.0 U,
1.5 U), three concentrations of template DNA (10 ng, 25 ng, 50 ng) and 10 pmole
primer in a volume of 25 µl. PCR was carried out using Thermal Cycler (Bio-Rad,
USA), PCR conditions that gave better amplified DNA profile .were determined and
presented below.
PCR constituents optimized for RAPD and ISSR analysis
Component Stock Quantity (µl) Final concentration
RAPD analysis
PCR Buffer with MgCl2 (15mM) 10× 2.5 1×
dNTPs mix 10 mM 2.0 200 µM each
Taq DNA polymerase I U/µl 1.0 1 unit
RAPD Primers 250 pM 1.0 10 pmoles
Sterile DNase, RNase free water 17.5
Total 24.0
Template DNA 1.0 25 ng
ISSR analysis
PCR Buffer with MgCl2 (15mM) 10× 2.5 1×
dNTPs mix 10 mM 2.0 200 µM each
Taq DNA polymerase I U/µl 1.0 1 unit
ISSR Primers 250 pM 1.0 10 pmoles
Sterile DNase, RNase free water 17.5
Total 24.0
Template DNA 1.0 25 ng
For RAPD analysis, all PCR were carried out in a final volume of 25 ml
reaction mixture containing 25 ng template DNA, 200 µM each dNTPs, 2 mM
MgCl2, 10 pmoles primer, 1× Taq polymerase buffer and 1 unit of Taq DNA
polymerase. The Thermal Cycler was programmed to include a pre-denaturation
step at 94oC for 3 min, followed by 44 cycles of denaturation at 94oC for 1 min
Page 33
annealing at 37oC for 1 min and extension at 72oC for 2 min. The final extension
was made for 7 min at 72oC.
For ISSR analysis, all PCR reactions were carried out in a final volume of
25 µl reaction mixture containing 25 ng template DNA, 200 µM each dNTPs, 2 mM
MgCl2, 10 pmoles primer, 1 × Taq polymerase buffer and 1 unit of Taq DNA
polymerase. Initially, the amplification was performed in programmable gradient
Thermal Cycler with following programme: a pre-denaturation at 94 oC for 3 min
followed by 44 cycles of denaturation at 94oC for 1 min, annealing at gradient
temperature for 1 min and extension at 72oC for 2 min. The final extension was
made for 7 min at 72oC. The annealing temperature tested was 5oC above and
below the melting temperature (Tm) of the particular primer. PCR amplification at a
particular annealing temperature that gave better resolution, that temperature was
selected for optimum annealing temperature for that ISSR primer for final PCR
amplification.
3.4. Agarose gel electrophoresis

Agarose gel (1.5%) was prepared by mixing 4.5 g of agarose in 300 ml of
1×TBE buffer. The content was boiled in microwave oven till it completely
dissolved. During warming intermittent shaking was made (4-5 times) to prevent
formation of clumps of agarose. The molten agarose was kept for cooling up to 50-
60oC and then ethidium bromide (1 μg/ml) was added. The molten agarose was
poured into the clean, leveled casting plate containing 30 well combs and was kept
for solidification. The gel was transferred to the electrophoresis unit containing 1X
TBE buffer. The PCR reaction products were first mixed with 2.0 µl of loading dye
(details given below) and spun for a while before loading into the wells of the gels.
The medium range ruler (Bangalore Genei) was also loaded in first and/or last well
of the gel to serve as standard molecular weight marker for determining the size of
the amplified DNA fragments. The gel was run at 80 volts for 4 h. The run was
stopped when bromophenol blue dye had travelled 2/3rd length of the gel.
3.5. Gel documentation and photography

After electrophoresis, the stained DNA gel was visualized under UV light in
a Gel-Doc system and photographed. Fragment size of all amplification products
Page 34
were estimated from the gel by comparison with standard molecular weight marker
(Medium Range Ruler). DNA bands were scored as discrete variable using ‘1’ to
indicate presence and ‘0’ to indicate the absence of a band.
3.6. Analysis of molecular data

It is imperative to understand the different ways that the data generated by
molecular techniques can be analyzed before considering their application to
diversity studies. Two main types of analysis are frequently done
 analysis of genetic relationships among samples,

 calculation of population genetics parameters, in particular diversity and its
partitioning at different levels.
The analysis of genetic relationships among samples starts with the
construction of a matrix specifying the character-state of each marker for each
sample. A sample will usually be DNA from an individual. Marker states may be
binary, as in the presence or absence of RAPD bands or restriction sites (as
revealed by RFLPs and related techniques). This sample × marker matrix of
character-states is then commonly used to construct a sample × sample matrix of
pair-wise genetic distances (or similarities). There are several different ways of
calculating the genetic distance (or similarity) between two samples on the basis of
the differences between them in the states of a set of genetic markers, but a
commonly used index is Nei’s genetic distance (D) or Jaccard’s similarity
coefficient (S). There are two main ways of analyzing the resulting distance (or
similarity) matrix and displaying the results. One is to use Principal Coordinate
Analysis (PCA) to produce a 2- or 3- dimensional scatter plot of the samples such
that the geometrical distances among samples in the plot reflect the genetic
distances among them with a minimum of distortion. Aggregations of samples in
such a plot will reveal sets of genetically similar material. Another approach is to
produce a dendrogram (or tree-diagram) linking together in clusters samples that
are more genetically similar to each other than to samples in other clusters.
Clusters are linked to each other at progressively lower levels of similarity until all
the samples being analyzed are included in a single cluster. Such Cluster Analysis
may proceed according to a range of different algorithms, but some of the more
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widely used ones include Unweighted Pair Group Method with Arithmetic Averages
(UPGMA), Neighbour-Joining Method and Ward’s Method. Both PCA and cluster
analysis are so-called ‘phenetic’ methods in that they are based on measures of
overall distance or similarity among samples.
However, there is another, philosophically quite distinct approach to the
analysis of genetic relationships, referred to as ‘cladistics’. Cladistic analysis also
begins with the sample × marker character-state matrix, and also results in
dendrograms, though these are sometimes called cladograms to distinguish them
from the phenograms of cluster analysis. The difference is that two samples are
placed together in the same cluster (or clade) of a cladogram not on the basis of
high genetic similarity between them calculated from all markers taken together,
but because they share a particular state of a given marker (or markers). The two
approaches are also sometimes distinguished as ‘distance’ and ‘character-state’
respectively. Because it is possible to generate many cladograms from a single
dataset, due to conflicts among characters, so-called parsimony approaches are
used to choose among them. A most-parsimonious cladogram is one that requires
the least number of character-state changes. There is a wide range of parsimony
algorithms, each with its own data requirements and assumptions. Some require
that the polarity of character changes be known, i.e. which character states are
ancestral and which derived. Cladograms are reconstructions of phylogenies.
RAPD data, because of uncertainty over the identity of bands, is not usually
thought suitable for this kind of analysis.
Turning now to the measurement of genetic diversity and genetic structure
(among and within populations), the F-statistics are commonly employed.
Estimates of these statistics are based on allele frequencies, and the most
appropriate molecular data for such statistical analyses are clearly those in which
allele frequencies can be determined directly, such as RFLPs, STMS and
sequence haplotypes. Of these, sequences and restriction site data are unique
among molecular markers in providing both frequency and phylogenetic
information. Nevertheless, suitable statistical treatments are also available for
dominant markers such as RAPDs.
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CHAPTER-4:
Techniques for virus testing and elimination
A large number of viruses infect horticultural, plantation and forest plants

which are being micropropagated by the tissue culture industry. Most important of
these are aphid-transmitted poty-, cucumo- and luteoviruses, whitefly transmitted
geminiviruses, thrip-transmitted tospoviruses, mealy bug transmitted ilarviruses.
Generation of virus-free planting material is an ideal strategy to confine these
viruses and also to facilitate the movement of materials across the domestic and
international boundaries.
Tissue culture is a useful approach for generating virus-free planting
material. In order to minimize the risk of inadvertent propagation of virus infected
plants and introduction of somaclonal variability, the tissue culture raised plants
need to be thoroughly indexed for freedom from viruses and checked for quality.
Careful indexing based on recent biotechnological methods such as imunoprobes,
nucleic acid probes and polymerase chain reaction (PCR) would ensure phyto-
sanitary safety during the movement of planting materials. Similarly, molecular
testing will ensure quality control.
The National Facility for Virus Diagnosis and Quality Control of Tissue
Culture Raised Plants (NFVD & QC), is aimed at providing virus testing and quality
testing support to the tissue culture industry to ensure that only (a) virus-free
mother plants are used for micropropagation, and (b) virus-free and quality tested
tissue culture plants are supplied to the growers.
Important viruses in Forest trees are
Bamboo : Bamboo mosaic potexvirus
Eucalyptus :Little Leaf
Poplar : Poplar decline potyvirus
Poplar mosaic carlavirus
Poplar vein yellowing mucleorhabdovirus
Page 37
4.1. Virus testing
Immuno-diagnosis is the most useful technique using polyclonal and
monoclonal antibodies raised against viruses. Polyclonal antibodies are now
available in the country for various groups of viruses like potyviruses, tospoviruses,
tobamoviruses, potexviruses, luteoviruses, badnaviruses, closteroviruses, etc.
These polyclonal antibodies have been very useful in the specific identification of
viruses and their strains, and for plant virus diagnosis. The technology for the
production of monoclonal antibodies is also available in our country, but their
production has not caught up to the desired level for plant viruses due the lack of
adequate facilities.
Various immunological and molecular techniques like enzyme linked
immuno sorbent assay (ELISA), immuno-electron-microscopy, Dot-immunobinding
assay (DIBA), Western blotting, immuno-fluorescence, nucleic acid hybridization
using radio-labeled probes and PCR have been used successfully for the diagnosis
of virus infections in a variety of plant species. Of these techniques, ELISA,
immuno electron microscopy, dot-blot and PCR are the most sensitive and reliable
techniques which can be very effectively used for the purpose of diagnostic
services to tissue culture industry in the country.
For nucleic acid hybridization, c-DNA probes are also available for the
Gemini, poty-, tobamo-, and badnaviruses.
4.1.1. Electrophoresis and direct electron-microscopy.

The viruses for which diagnostic probes (antibodies and cDNA probes)
have been developed are known to infect a variety of plant species like: banana,
brinjal, cassava, citrus, French bean, garlic, gladioli, groundnut, lilies, mungbean,
onion, rice, soybean, sugarcane, tobacco, tomato, urdbean, ornamental and forest
plants etc.
Microsatellite primed-Polymerase Chain Reactions (MP-PCR) were used to
study genetic fidelity of regenerated shoots of Eucalyptus, Populus and Coffee
species. Polymorphisms could not be detected in bread wheat genotypes using 23
simple sequence repeats indicating the inadequacy of the molecular analysis. In
Populus deltoides six of the off-types were reported through RAPD analysis of 32
Page 38
randomly selected plants from a population of 500 individual plants. The plants
were derived from nodal segments.
4.2. Virus indexing using molecular techniques

There are many published protocols for detection of banana viral
pathogens. Serological tests (ELISA) are based on antigen-antibody reaction in
which the viral coat protein acts as antigen and specific antibody raised against
coat protein serves as antibody for immunological tests. In case of nucleic acid or
viral genome based techniques, PCR, RT-PCR and nucleic acid spot hybridization
(NASH) have been standardized and are widely used in virus detection. At National
Research Centre for Banana (NRCB), ELISA, PCR and NASH have been
standardized and are being used to detect viruses in banana mother plants and
tissue culture derived plants
4.2.1 Protein based methods

Detection of plant viruses by serological assay is being widely practiced
since the discovery of enzyme-linked immunosorbent assay (ELISA) technique by
Clark and Adams (1977). Various forms of serological assays are currently
available for all seven known banana viruses. ELISA is a sensitive and efficient
practical assay for banana virus indexing, when suitable antisera are available.
Specific polyclonal and monoclonal antibodies are available for BBTV, BSV, CMV
and BBrMV (Diekmann and Putter, 1996). ELISA tests with monoclonal antibodies
(MABs) have been commonly used for the accurate detection of BBTV. However,
Page 39
ELISA is of limited use when very low concentrations of BBTV occur in infected
plants. Further, viruses belonging to geminiviridae are less immunogenic and yield
low titre polyclonal antisera. Geering and Thomas (1996) developed triple antibody
sandwich ELISA for virus indexing of BBTV which is preferred method when
compared to dot immunobinding assays and amplified ELISA. Other variants have
been introduced for better detection of BBrMV, BSV, BBTV and CMV.
4.2.2 Nucleic acid or viral genome based methods
4.2.2.1 Nucleic acid hybridization

New techniques based on viral nucleic acids are becoming popular
and wide spread as molecular diagnostic tools. Nucleic acid hybridization of
DNA or RNA probes has the advantage of being able to detect the nucleic acid of
the virus in both single-stranded and double-stranded replicate forms. Radioactive
isotopes like P32 are used for labelling nucleic acid probes and the signal is
detected by autoradiography. In recent years, non-radioactive labeling and
detection using biotin/ streptavidin or Dig-oxigenin (DIG) systems have been
widely used for many viruses (Dietzgen et al., 1994; Mas and Pallas, 1995; Mas
et al., 1993). When large numbers of samples are to be tested in a short time,
dot blot hybridization or nucleic acid spot hybridization (NASH) is the appropriate
technique. In this technique, the viral genomes are detected from crude or
purified samples after hybridizing with labelled specific probes. It is easy to
send the samples after spotting than sending live tissues to testing centres
as they get spoilt in transit. NRCB has developed NASH technique for BBTV
which is equally sensitive as PCR and the technique has been applied for
detection of BBrMV, CMV and BSV.
4.2.2.2 Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) based detection systems are now
available for all banana viruses. Xie and Hu (1995) used PCR for detecting
Hawaii isolates of BBTV and it was 1,000 times more sensitive than ELISA or dot
blots with DNA probe. A simple, single-step plant tissue preparation protocol to
reduce plant inhibitory factors interfering with PCR and suitable for the detection
of BBTV in corm, leaf and root tissues has been developed. Galal (2007)
Page 40
used PCR for detection of BBTV in banana samples exhibiting characteristic
symptoms of BBTV, and also from the viruliferous banana aphid. Selvarajan et
al. (2007b) has also developed PCR based method to detect Indian isolates of
BBTV. BBrMV was detected by RT-PCR in total nucleic acid extracts from
infected plants, using specific or degenerate potyvirus group primers. PCR
based diagnostic assay for BSV is difficult as its genome integrates with banana
host genome. Complete sequences for many species of BSV have become
available for designing primers for detection. Specific PCR are now available
for some BSV strains, but there are additional uncharacterized isolates not
detected by PCR assay, which show great genomic and serological
heterogeneity.
Cucumber mosaic virus (CMV) causing mosaic and chlorosis of
banana has been detected by RT-PCR amplification of coat protein gene
(Aglave et al., 2007). Polyvalent degenerate oligonucleotide RT-PCR was used to
detect Banana mild mosaic virus (BaMMV) and Banana virus X, two flexiviridae
infecting Musa spp. BVX could be detected using species specific primers
whereas PDO-inosine containing primers were found well suited for detection
of BaMMV (Teycheney et al., 2007).
4.2.2.3 Multiplex PCR and Immuno-capture PCR

Multiple species or strains of viruses are detected simultaneously in
a single PCR reaction by combining oligonucleotide primers specific for
different viruses, a technique termed as Multiplex-PCR. In this technique,
DNA fragments to be amplified are of different lengths and there should
not be any cross reactivity between different viral species targeted.
Immuno-capture PCR (IC-PCR) method combines both serology and PCR
techniques. In IC-PCR, the virus particles are first trapped onto a specific
antibody bound to a surface. The trapped virus particles are disrupted and
the viral nucleic acid released and amplified by PCR or RT-PCR. This method
is especially useful in concentrating virus particles from plant species where
virus titre is low and compounds that inhibit PCR are present. Sharman et al.
(2000) standardized multiplex-immuno-capture PCR (M-IC-PCR) for simultaneous
detection of BBTV, BBrMV and CMV in banana. Simultaneous detection of Indian
Page 41
isolate of BBTV and BSV with duplex PCR and BBrMV by multiplex-PCR has
been reported (Selvarajan et al., 2004a). Salvarajan et al. (2004b)
developed IC-PCR technique of BBTV. Immunocapture RT-PCR has also been
used to detect BaMMV (Teycheney et al., 2007).
4.2.2.4 Real Time PCR

Of late, a novel real-time quantitative PCR technique, Real Time PCR,
has been developed for the detection and quantification of plant viruses. It is
more sensitive, reliable and specific than PCR. It reduces the risk of cross-
contamination, obviates post PCR manipulations, provides higher throughput,
and enables quantification of virus load in a given sample. However, this
technology requires more expensive equipment and reagents compared with
conventional PCR.
Rapid, reproducible and specific detection of episomal BSV from crude
extracts of infected plants by real time assay has been developed for large
scale screening. Primers have been designed to specifically amplify the episomal
BSVOLV sequence of 1,336 bp size to detect in real-time by a short fluorogenic
3’ minor groove binder DNA probe for 14 bp conserved BSV sequence. Due
to cost factor, this technology may not be feasible for small scale banana
tissue culture set up where small number of samples are handled.
4.2.2.5 Direct-binding PCR (DB-PCR)

This technique is based on the adsorption of viral particles to the
surface of a polypropylene micro-centrifuge tube. Direct binding PCR has
successfully been used at NRCB to detect BSV, BBrMV and CMV (Selvarajan
et al., 2007a). This technique has also been used to detect the episomal form
of BSV. This assay is cost effective and less time consuming.
4.3 Elimination of viruses

Virus affected planting material poses a major problem in propagation and
exchange of germplasm, and eventually in breeding and distribution of superior
genotypes. For many banana viruses, the nature of spread still remains unclear
and most of the field gene banks are severely affected. Meristem-tip culture,
Page 42
chemotherapy, electrotherapy and cryotherapy are some of the methods
applied for elimination of viruses in banana and other vegetatively propagated
crops. They are used either alone or, in most cases, in combination.
Kassanis (1950) first used high temperature treatment to eradicate virus from
potato tubers. Later, techniques involving thermotherapy or tissue culture and
frequently a combination of both have been successfully used to eradicate
viruses from infected plants (Walkey, 1980).
4.3.1 Meristem-tip culture

This technique involves the use of apical dome or shoot tip with a
few leaf primordia of the size less than 1 mm in length as the explant. In banana
and plantain, meristem-tip culture is considered to be the reference tool for virus
eradication. The application of meristem-tip culture to eradicate viruses was
initially based on the concept of meristem “immunity” towards viruses (Morel,
1948). Different researchers have shown that the probability of obtaining
virus-free plants is inversely related to the size of the meristem (Faccioli and
Marani, 1998).
4.3.2 Thermotherapy
Heat therapy has been used to successfully eliminate many viruses
from a variety of plant species (George, 1993). The heat treatment may be
done either in vitro or in vivo and is usually combined with meristem culture
for better results. CMV infected in vitro and in vivo plants of banana cv.
Williams BSJ (ITC 0570) were kept for one day in a growth cabinet under
artificial light with diurnal alternating periods (16 h light/8 h dark). Day
temperature in the growth cabinet was initially 28 ± 1 °C which was increased
at 2 ± 1 °C per day until 40 ± 1 °C was obtained. Plant material was kept at this
temperature for 4 weeks, with night temperature at 25 ± 1 °C. Meristems
(domes with four-leaf primordia) were then excised and transferred to MS
medium. The percentage of healthy plants regenerated after a combination of
meristem culture and thermotherapy was 38 % and 70 % when meristems
were excised from in vivo or in vitro plants, respectively. Berg and
Bustamante (1974) observed that heat treatment (35-43 °C for 100 days)
Page 43
performed on rhizomes (2.5 cm square and 5-7.5 cm long) in conjunction with
meristem culture was inefficient for cleaning commercial banana cultivars.
However, 56 plantlets out of 73 regenerated by culturing meristems of heat-
treated rhizomes did not produce symptoms when extracts were used to
inoculate indicator plants. CMV eradication was also achieved by Gupta
(1986) in approximately 100 % of the regenerated plants when using
meristem culture in combination with a two-week heat therapy (38°C-40°C).
4.3.3. Chemotherapy
Chemotherapy, either alone or in combination with other techniques, is
becoming increasingly available as a virus elimination tool (George, 1993). Anti-
viral chemicals may be either sprayed on plant or incorporated into tissue
culture media. Often, a chemical therapy is followed by meristem culture. Antiviral
substances such as acyclic adenosine analogue [(RS)-9-(2, 3-dihydroxypropyl)
adenine [(RS)-DHPA] and ribavirin (1-b-D-ribofuranosyl-1,2,4-triazole
carboxamide; Virazole®) have been used. Meristems excised from CMV-
infected banana were grown in a culture room (standard conditions) for 3
months on MS medium to which 50 mg/l of Virazole or (RS)-DHPA was added
(Helliot et al., 2004). Concentrations of chemical compounds used for
chemotherapy assays were determined after the phytotoxicity tests. The
percentage of healthy plantlets regenerated from Virazole treated highly
proliferating meristems reached 29 % while only 2% of plantlets were found
virus-free when regenerated from (RS)-DHPA treated highly proliferating
meristems.
4.3.4 Electrotherapy
Electrotherapy assays were carried out either on infected in vivo or in vitro
plants (Hernandz et al., 1997). Pulses of 15 V were applied for 5 min to 2-
3 cm long explants containing apical meristem. The meristems were then
excised and placed on an MS culture medium. The efficiency of electrotherapy
in producing virus-free regenerants from BSV-infected banana plants (cv. W.
Bungulan) was 40-80 %. In case of CMV, the preliminary percentage of healthy
plantlets regenerated from electrically treated explants obtained from in vivo
Page 44
plants reached 11% while no plantlet out of 27 tested was found to be
virus-free when explants were obtained from in vitro plants ( Helliot et al., 2004).
4.3.5. Cryotherapy
Cryotherapy of shoot tips based on cryopreservation techniques is a
new method for pathogen eradication. Cryopreservation refers to the storage of
biological samples at ultra-low temperature of liquid nitrogen (−196°C), and is
considered an ideal means for long-term storage of plant germplasm.
Helliot et al. (2002) reported the utilization of cryopreservation for
eradication of CMV or BSV from Banana. Plants of cv. Williams (AAA, Cavendish
subgroup) were mechanically infected with CMV or naturally infected with
BSV, and proliferating meristems were produced from the infected plants.
Excised meristematic clumps were cryopreserved through vitrification using
PVS-2 solution. The health status of regenerated in vitro plants was first
checked by means of ELISA. The putative virus-free material was subsequently
tested a second time following greenhouse acclimatization. The frequency of virus
eradication for CMV and BSV following cryopreservation was 30 % and 90 %,
respectively. In comparison, the frequency of virus-free plants regenerated
directly from highly proliferating meristems, corresponding to a spontaneous
eradication rate, was 0 % and 52 % for CMV and BSV, respectively. The
conventional meristem culture resulted in 0 % CMV-free plants and 76 % BSV-
free plants, while the cryoprotective treatment resulted in 2 % CMV-free plants
and 87 % BSV-free plants.
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CHAPTER 5:
Micropropagation for Quality Banana Plantlets
Micropropagation is the practice of rapidly multiplying stock plant

material to produce a large number of progeny plants under aseptic
conditions using modern plant tissue culture methods. Application of
micropropagation in banana has the following advantages:
a. Rapid multiplication
The rate of multiplication in banana is restricted to 5-20 suckers per plant
during its growth period, which makes it difficult to obtain sufficient amount of
planting material of a clone of choice. Micropropagation facilitates production of
large number of plantlets/unit time, thus helping in rapid introduction and
dissemination of new varieties.
b. Requirement of limited mother stock

The rapid multiplication technology ensures that limited number of
mother plants are required for raising large number of progeny plants.
These few mother plants can be maintained with required care at a limited
cost.
c. Product uniformity
Being a vegetative reproduction method, micropropagation results a high
degree of genotypic and phenotypic uniformity of the progeny plants. The
limited variation seen sometimes can be overcome by following appropriate
micropropagation, genetic fidelity testing roguing protocols.
d. Season independent production

In conventional field propagation, the production of suckers is highly
season dependent and, hence, availability of planting material in a given season
is often a limiting factor. The planting season in most of the banana production
areas starts with the onset of monsoon, which creates a heavy demand for the
planting material often leading to supply of substandard material. Using
micropropagation, the production of planting material can be achieved as per
needs.
Page 46
e. Agronomic advantages
Micropropagated plants exhibit uniform growth and maturity enabling
one time harvesting. The once over harvest provides a gain of 60-70 days
which allows the farmers to take a short duration legume crop that adds to the
income and soil fertility. It also saves on labour and energy for
transportation. These are major concerns of the growers.
f. Production of disease free planting material

Using tissue culture, it is possible to develop planting material which is free
from sucker borne diseases and pests. Use of healthy planting material
complemented with integrated pest management program is the key to a good
crop stand in field.
g. Plant exchange
Production of plants in test tubes facilitates safe movement and easy
handling of germplasm between laboratories within and across countries.
h. High returns
Since the micropropagation based progeny is genetically and
phenotypically similar to the mother plant, which is often a superior selection,
the yield and returns are expectedly higher.
3.1 Shoot tip culture

The earliest reports of in vitro culture of bananas came from Taiwan in
the 1970’s. Till date, protocols have been standardized for in vitro propagation
of a wide range of Musa species and cultivars belonging to various ploidies and
genomes.
Shoot tips can be extracted from the pseudostem, suckers, peepers,
lateral buds or even small eyes which contain a shoot meristem (Jarret et al.,
1985; Vuylsteke and De Langhe, 1985). Though all of them behave similarly
under in vitro conditions, peepers and sword suckers are preferred because of
their ease of handling and the minimum damage caused to the parent stool
during their removal. It is always better to collect the explants from flowering
plants so as to ascertain their trueness to type.
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The steps followed for production of micropropagation based banana
planting material are:
Selection of mother plant and establishment of mother block nursery,
 Virus indexing of mother plant nursery,

 Culture initiation,
 Culture proliferation,
 Rouging at various stages of proliferation,
 Rooting and primary hardening accompanied by roguing,
 Secondary hardening accompanied by roguing,
 Fidelity testing and virus indexing at various stages of mass
multiplication.
5.1.1. Selection of mother plants and establishment of mother nursery

Selection of proper mother plants and standard requirements for a
mother block nursery is as follows
Mother plant should be
 Healthy, true to type and free from diseases and pests, especially virus
diseases.
 The male flowers buds should be retained to check the presence of virus
diseases (male flower buds exhibit symptoms of late infection of viruses like
BBTV and BBrMV).
 Mother plants should be raised under roofless insect proof shade net with
sufficient height.
 Mother nursery must be located away from other banana plantations with an
isolation distance of 500 m to maintain purity and to avoid spread of virus
diseases.
 Mother plants should be grown under very good management conditions so
as to facilitate the true expression of traits.
 Individual plants should be tagged with a master code number so that
theplantlets developed could be traced back to the mother plant.
 Pedigree record and source of each mother plant should be maintained and
catalogued.
Page 48
 Once indexed, the mother suckers can be maintained in field or concrete
rings with frequent decapitation to facilitate production of more axillary buds.
They also serve as explants for culture initiation.
5.1.2 Virus indexing of mother plant nursery

Detailed indexing procedures for banana viruses are presented in
previous chapter. Indexing should be carried out primarily for four viruses, namely,
BBTV, BSV, BBrMV and CMV, and should be done twice during crop duration,
at 6 months stage and at fruiting. If found infected, the entire clump comprising,
suckers along with underground mother corm should be removed and
destroyed.
5.1.3 Selection of superior initial planting material

Choice of explant is vital for which purpose well maintained mother plants
should be selected. Sword suckers should be healthy and not less than 60-80
days of age while the growing meristem should be of 1.0 cm 3 in size.
5.2 Culture medium

Success of in vitro culture depends largely on the choice of nutrient
medium, including its chemical composition and physical form (Murashige,
1974). Several media formulations has been reported for banana shoot tip
culture but nearly half of them are modified MS media (Brown et al., 1995).
Other popular media include (Gamborg et al., 1968), SH (Schenk and
Hildebrant, 1972), (Chu et l., 1975), and Linsmaier and Skoog (LS) (Linsmaier
and Skoog, 1975) media. The culture media vary in both type and
concentration of the components, but all have similar basic components of
growth regulators, nitrogen, carbohydrates, inorganic macro and micronutrients,
vitamins and organic additives.
Generally, the cultures are established on a separate initiation
medium, which has a lower concentration of cytokinin than the multiplication
medium to which the cultures are subsequently transferred. The composition
of initiation, multiplication and rooting media used at the National Research
Centre for Banana, India (NRCB) is given below.
Page 49
Culture Media Used at Different Stages of Banana Micropropagation (for one litre)
After autoclaving, the culture medium is stored in a clean dust free

chamber for 1-2 days before use in order to check for any contamination. Bacterial
contamination may be observed, particularly during the rainy season. Use of
Cefotaxime in the initiation and subsequent subcultures helps to overcome even
latent bacterial contaminations.
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5.3 Culture initiation
The sword suckers of 2-3 months are removed from healthy disease
free mother plants for shoot tip culture. The suckers are cut to expose the
shoot tip of 10 cm3 and cut further to about 3 cm diameter and 5 cm
length. The explant should be carefully cut to avoid injury to the growing
meristem. The shoot tips are washed in tap water and transferred to a
container with 0.1 % mercuric chloride for 10 min and then to 0.1 % cetrimide.
Then the shoot tips are washed thoroughly under running tap water to remove
all traces of the chemicals. Using sharp sterile blade, one or two outer juvenile
leaves and the corm base are trimmed out. Afterwards, the shoot tips are
washed three times in sterile water in aseptic condition (under laminar air flow)
disinfected with 5 % sodium hypochlorite and later with 0.1 % mercuric
chloride each for 15 minutes. To avoid bacterial contamination, use of
Cefotaxime (0.1 %) in the initiation medium is in vogue in some laboratories.
Surface sterilized shoot tips are washed three times using sterile water.
The outer surface of explant exposed to sterilizing agent is removed and the
explants trimmed using surgical blade (No. 22) to bring the final size to about
3-4 cm length and 1-2 cm diameter. The explants are inoculated under sterile
conditions in 30 ml of initiation medium in a 250 ml glass jar container. pH is
usually maintained at 5.8, which is prone to changes over culture duration. The
optimum incubation temperature should be in the range of 24-26 °C. Generally
the light intensity is maintained at 1,500-3,000 lux. Higher levels of 3,000-10,000
lux during later stages improve the survival rate of plantlets upon transfer to soil.
Initially, the cultures are maintained at 16 h light/8 h dark cycle and once
after rooting they are shifted 14 h light/10 h dark cycle.
Decapitation and wounding of shoot tips are carried out to
overcome apical dominance and to encourage auxillary bud proliferation. But
injuring the apical bud through transverse sections, either four or eight cuts,
is a much preferred method. Injuring the explant encourages more production
of phenols, but it can be kept at minimum using antioxidants like ascorbic acid.
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5.4 Culture proliferation
First subculture is done after 20-25 days of initiation when the explants
turn green in colour. The cultures are first checked for contamination, in general
symptoms of fungal contamination appear within one week and bacterial
contamination symptoms like change of medium colour and texture or visible
colonies appear within one week to one month. For sub-culturing, the outer
dead tissue from the base of explant is removed and one or two leaf bases
are peeled till the fresh meristematic tip gets exposed. The apical meristem
is cut with two gentle cross incisions and the explant is transferred to subculture
medium. During 20-25 days after the first subculture, the central meristem
produces clusters of proliferating buds and one to three axillary buds get
regenerated from the basal parts of explants around the central apical
meristem. The number of axiliary buds developed during first and second
subculture range from 1 to 5 depending on genomic constitution of the variety. In
general, diploids like Matti, Anaikomban and Senna Chenkadali produce more
buds than commercial cultivars. Among the latter, the number of buds
produced during subculture is high in Cavendish (Robusta, Grand Naine –
AAA genome) group followed by Plantain (Nendran – AAB genome) and
Monthan (ABB genome) types.
Subsequent subculture is done by trimming the tip of emerging
axillary buds and removal of dead tissue at the base of explant by gentle
scratching. Clusters of proliferating buds develop during third and fourth
subculture. For further subculturing, the explant is cut into three to four pieces
and each slice with two to three proliferating clusters is inoculated to individual
culture bottles. This subculture cycle is repeated at 3-4 weeks interval to
increase the proliferation rate. During fourth and fifth sub-cultures, a single
clump contains about 15-25 proliferating shoots. After 5-6 sub-culture cycles, the
proliferated buds are transferred to rooting medium containing IBA and
activated charcoal. After a month, the rooted plantlets are ready for hardening.
To minimize somatic variation, the sub-culturing is restricted to a maximum of
seven cycles when each bottle contains 25-30 plantlets with well developed
shoots and roots.
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Experiments have demonstrated that proliferating shoots can be
transferred to polybags (10-20 cm size) having rooting media under green house.
This reduces cost and enhances better establishment. Polybag provides
enough space for plant growth and natural light enhances the process of
hardening.
5.5 Hardening
Once the plantlets are ready for shifting outside the laboratory, they are
carefully acclimatized to adapt to the green house and later to least protected
field conditions. During hardening, the plantlets undergo physiological adaptation
to changing external factors like water, temperature, relative humidity and
nutrient supply.
The plantlets from culture vessels/bottles are moved from the laboratory to
a room at ambient temperature and kept open for 4-6 days. Later they are
shifted to green house for primary hardening where they are first gently washed
free of agar medium. This is important as sucrose in agar encourages
microorganisms. 8 cm shoots with 3-4 ramified roots are planted in individual
micropots in a protray. In places where weather is conducive (24-26 °C
temperature and more than 80 % humidity), the plantlets are hardened for 4-
6 weeks in mini-sand beds. During this period, 90-95 % humidity is maintained
for the initial 6-8 days under diffused light. The humidity is slowly reduced to
70 %, light intensity raised to normal and temperatures brought to 26 °C by the
end of 6 weeks.
Structures used for primary hardening vary with the climatic conditions.
These can be highly sophisticated with UV-stabilized polysheet covering,
multiple misting options, thermal shade net and auto-monitoring of light
intensity, temperature and humidity. On the other hand, the structures can be
simple with polycarbonate roofing, shade net on all sides with fogger facilities.
Temperature, RH and light intensities are monitored manually using thermometer,
hygrometer and lux meter, respectively.
Planting media for primary hardening range from sieved sand
augmented with nutrition to mixtures of cocopeat and soilrite with fine sand in
equal proportions. NPK is provided in liquid form on weekly basis.
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5.6 Secondary hardening
After primary hardening for 5-6 weeks, the plantlets are transferred from
micropots to polybags. Base substrate is generally soil and sand along with
low cost materials like coir pith, sawdust or rice husk. Organic manure is
either in the form of farm yard manure or poultry manure. In Maharashtra,
India, Press mud, a byproduct of sugar factories, has been found to provide
best substrate for secondary hardening along with soil (Vasane et al., 2006).
Plantlets from micropots are, dipped in fungicide solution (0.1% bavistin) and
planted in polybags containing suitable substrate. Initially, these are
maintained in low light intensity shade nets and 70 % RH. The plants are
hardened by gradually increasing the light intensity and reducing RH (40 %).
After 5-6 weeks, the plants become ready for field planting having 3-5 well
developed leaves and a good mass of fibrous roots.
During both primary and secondary hardening, the stocks should be
rouged for variants at weekly intervals. These could include vegetative deformities
like dwarfism, leaf variegation, rosette foliage and leaf crinkiness. Other
precautions to be followed are:
 The rooting media should be completely free from pathogens.

 Water used for irrigating the plants should be free from pests
and pathogens.
 Sample plants from each batch should be randomly virus indexed (at
least 10 plants from each batch/explant)
 While shifting primary hardened plantlets, two longitudinal cuts should
be given to the micropots to facilitate further corm growth.
5.7 Manuring and plant protection in nursery

Plantlets should be 2-3 weeks old before any fertilizer is applied. 100 ml
water containing 0.5 g urea, 2 g superphosphate and 1 g muriate of potash can
be applied per plant. The manuring is repeated by doubling the dosage after three
weeks. Spraying of commercially available micronutrient mixtures during sixth
week helps in better establishment both in nursery and field. Strict sanitary
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measures are adopted in the nursery to avoid the risk of damage by pests
and diseases either through substrate or irrigation water.
5.8 Field planting and initial management

20-30 cm tall plants with 3-5 broad leaves are ready for field
planting. At the time of planting, 10g of Carbofuron is applied per plant.
Watering is done soon after field planting as young micropropagated plants are
sensitive to dry weather and heat. Since these are also highly susceptible to
bacterial rot (Erwinia rot), within 3 days of planting the soil around the plants
is drenched with 500 ml of 0.1 % Emisson (Methyl Ethoxy Mercuric Chloride).
Recommended package of practices is strictly followed to achieve successful
field establishment and subsequent vigorous growth. An ideal tissue culture
raised plant should:
 be 30 cm in height and have a pseudostem circumference of 5.0-6.0

cm after 60 days of total hardening;
 have 4-5 photosynthetically active leaves and inter-foliar space must
be not less than 5.0 cm;
 have approximately 25-30 more than 15 cm active roots at the
end of secondary hardening;
 be free from any visual symptoms of leaf spot, pseudostem rot and
physical deformations;
 be free from root pathogens like Erwinia, nematode lesions and root
knots.
Random checking of roots is essential to ensure health of plantation.
5.9 Testing for genetic fidelity and virus infection

Virus indexing and genetic fidelity testing are important to produce
good quality disease free planting material using tissue culture technology.
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L i st o f I S S R p ri m er s u sed f o r g en et i c f i d el i t y t e st i n g
Shoot tip cultures preserve genetic stability much better than callus or cell
suspension cultures, yet somaclonal variation seems to be widespread
among plants regenerated from banana shoot tip cultures. Commercial varieties
like Robusta, Grand Naine, Dwarf Cavendish, Champa are highly susceptible to
these variations and the off-types number up to 74 %. High level of variation is
not desirable as it defeats the purpose of clonal reproduction and majority of
the off-types are agronomically inferior to the parental clone. Banana and
plantains have a flexible genetic make and its genetic stability under cultive is
strongly influenced by external factors like growth regulators, duration of culture
etc. Mild stress under in vitro results in reverting of the clone to its parental
type. For example, Robusta, a Cavendish clone frequently reverts to its original
type Dwarf Cavendish which is not acceptable for its low stature and poor yield.
Hence, genetic fidelity testing using preferably molecular marker techniques is
essential to ensure the supply of true to type quality planting material. At NRCB,
besides phenotype PCR technique with ISSR markers is being successfully used
for screening off-types in banana.
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Figure summarizes the various stage in micropropagation of banana.
Using the protocol, more than 10,000 plants are expected from a single
explant at the end of 320 days
Projected multiplication rate of banana under of tissue culture from a
single explant
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CHAPTER 6:
National Certification
ertification System for Tissue Culture Plants
The Department of Biotechnology (DBT), Ministry of Science &

Technology, Government of India, as authorized under section
section-8
8 of Seeds Act,
azette notification dated 10 th March
1966, vide gazette arch 2006, is the Certification Agency
for the purpose of certification of the tissue culture raised propagules up to
laboratory level and to regulate its genetic fidelity as prescribed. The Certification
Agency (DBT) is responsible for implementing the Na
National
tional Certification System for
Tissue Culture Plants (NCS
(NCS-TCP) in the country.
ountry. National Certification System for
Tissue Culture Plants (NCS
(NCS-TCP)
TCP) is the unique system developed by DBT to
support the plant tissue culture industry.
6.1. Structure of National Certification System for Tissue Culture Plants

(NCS-TCP)

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The need for a certification programme for the tissue culture plants is
imperative since inadvertent micropropagation of virus infected plants will not only
result in its poor performance, but also in undesirable spread of viruses wherever
such plants are grown. Also, failure to use prescribed standard protocols will result
in variations in the plants produced. The most deleterious variants in tissue culture
raised plants are those that effect yield, genetic fidelity and carry infection of
viruses, and other fastidious pathogens, which are difficult to diagnose. This is an
area of great concern, and requires a well-structured system be put in place to
provide support to the tissue culture industry for the commercialization of tested
virus free and high quality planting material.
6.1.1. Tissue Culture Certification Agency (TCCA) of (DBT):

TCCA is responsible for implementing the National Certification System for
Tissue Culture raised Plants (NCS-TCP) in the country. The Certification Agency is
overall responsible for developing standard tests, production protocols/guidelines
and manuals.
6.1.2. NCS-TCP Management Cell (NMC):

NMC has been established by the Tissue Culture Certification Agency
(TCCA) at Biotech Consortium India Limited (BCIL) for assisting DBT in
Accreditation of Test laboratories for virus diagnosis and genetic fidelity/ uniformity
testing of tissue culture raised plants and Recognition of Tissue Culture Production
Facilities.
6.1.3. Referral Laboratory (RL):

The DBT has designated Referral laboratory(ies) for virus
diagnosis/genetic fidelity testing of tissue cultures plants.
 Referral Center for Virus Diagnosis – Indian Agriculture Research Institute

(IARI), New Delhi
 Referral Centers for Genetic Fidelity/ Uniformity – National Research Center
on Plant Biotechnology (NRCPB), New Delhi
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The Referral Laboratory will not involve in routine Virus diagnosis/Genetic
fidelity/ uniformity testing of tissue culture raised plants, but undertakes random
testing of samples.
6.1.4. Accredited Test Laboratories (ATLs):

It is responsible for testing the Tissue Culture material for Virus diagnosis
and Genetic fidelity/ uniformity, for the purpose of certification. Prepares a Test
Report based on tests conducted in conformity with the
standards/protocols/guidelines. Each Accredited Test Laboratory (ATLs) is
authorized to issue the Certificate of Quality for the Tissue Culture Plant (CQ-TCP)
along with certification label on behalf of the Tissue Culture Certification Agency.
6.1.5. Recognized Tissue Culture Production Facility:

Recognized Tissue Culture Production Facility should adopt Standard
Operating Procedure (SOP) and maintain all relevant records. Recognition of
Tissue Culture Production Facility is granted for a period of TWO YEARS
thereafter it would be re-assessed for “Renewal of Recognition”.
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6.2. Process for Accreditation:
6.3. Standard Guidelines/Parameters for Production and Certification of

Tissue Culture Raised Plants
6.3.1. Testing of Mother Plant Tissue/Stock Culture

Virus indexing of stock culture could be done from Accredited Test
Laboratory (ATL) or any other reputed organization but certification of tissue
culture raised plant testing of material for freedom from virus would be only done
by Accredited Test Laboratories (ATLs) under NCS-TCP for recognized Tissue
Culture Production Facility (TCPFs).
Prior to the certification of tissue culture raised plant “Mother Plant
Tissue/Stock Culture(s)” need to be indexed for all known virus listed under NCS-
TCP. For the virus indexing of Plant Tissue/Stock Culture(s) companies need to
request the nearest Accredited Test Laboratories (ATL) through “Intimation form for
Virus Indexing of Plant Tissue/Stock Culture(s)” along with self addressed
envelope. The above request should reach the ATL two weeks before the sample
is (are) being sent. The ATL will examine the Annexure-1A and if found complete
in all respect it will intimate the applicant, regarding requirements for sending the
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samples. Guidelines for dispatch of material, sample size, quantity of sample, fee
etc. will be provided. Subsequently after the receipt of acknowledgement from ATL
the companies need to send “Application for Virus Indexing of Plant Tissue/Stock
Culture (s)” (Annexure-2A) and samples along with prescribed fee.
Process Flow and Relevant Formats for Testing of Mother

Plant Tissue/Stock Culture
Intimation form (Annexure 1A) will be received from TC companies for Virus
Indexing of Plant Tissue/ Stock Culture(s) preferably at least two weeks before the
sample(s) received. ATL will acknowledge the intimation and inform the company
regarding fee to be submitted for testing`
Sample(s) will be received along with application (Annexure 2A) covering

detailed information of the samples to be tested and requisite fee. Each sample will
be assigned unique 20 digits sample registration number by ATLs.
The samples would be forwarded by in- charge ATL to technical person

with a job card
The laboratory technician (Virology) after testing will prepare a test report and
submit to Scientist concerned to verify and sign the test report
In case of dispute, the concerned ATL will forward the second sub-sample to
referral laboratory
ATL will maintain the whole record in master register for stock cultures
6.3.2. Testing of Mother Plants/Stock Culture

1) All mother plants/stock cultures must be indexed for all the viruses affecting
the plant species listed in NCS-TCP website.
a) Ideally individual mother plants/stock cultures should be tested.
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b) If the number of mother plants/stock culture is large, the samples from
batches consisting of a maximum of 10 mother plants/stock cultures may
be pooled for testing.
In such cases –
i. The tissue culture unit must maintain proper record of individual

mother plants/stock cultures of each batch, so that individual mother
plants/stock cultures or smaller batches could be tested, in cases where
the pooled samples are found positive for infection, so that only the
cultures from infected mother plant/stock culture are discarded.
ii. If testing is not done as envisaged in 1(b)(i), all the cultures

generated from the infected mother plants/stock cultures will have to be
discarded.
2). Virus testing can be done ATLs or any Govt. Institute or University having
facilities and expertise for virus testing.
6.3.3. Certification of Tissue Culture Raised Plants:

Only Recognized Tissue Culture Production Facilities will be eligible to
register for certification of plant tissue culture raised material. ATL would not
accept the sample of tissue culture raised plants for certification if the mother
plant/stock culture has not been indexed for respective batch of TC plants.
The Tissue Culture Production Facility will register its application for
Certification of Tissue Culture material with the nearest ATL through “Intimation
form for (Virus/ genetic fidelity) Testing for Batch Certification of Tissue
Culture Raised Plants” (Annexure-1B) along with self addressed envelope. The
above request should reach the ATL 2 weeks before the sample is (are) being
sent. The ATL will examine the Annexure-1B and if found complete in all respect it
will intimate the applicant (in two working days), regarding requirements requisite
fee for the testing.
The fees for batch certification of tissue culture raised plant and
“Application for (Virus/ genetic fidelity) Testing for Batch Certification of
Tissue Culture Raised Plants (Annexure-2B)” will be deposited at the ATL (The
details are at SOP for TCPUs and ATLs). An Annexure-1B and Annexure-2B
might be downloaded from NCS-TCP web site.
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The ATLs, will arrange for site inspection, receipt of samples, testing etc
as per prescribed format. On receipt of requisite fee ATL personal should visit the
hardening facilities of recognized tissue culture production facilities for collecting
samples for batch certification. The cost of the visit might be borne by the ATL
under travel head.
6.4. Certificate of Quality and Certification Label:

The ATL will generate the Test Report within the prescribed time frame
and based on the test report the Certificate of quality will be issued as per
prescribed norms. In addition, the concerned ATL will issue 10 number of
certification labels duly signed and stamped for affixing on the packages of
consignment of tissue culture plants as prescribed under certification standards
established by the Department of Biotechnology in accordance with provisions of
Seeds Act, 1966. Additional label will be provided by ATLs to TCPFs only on
written request from company without any charges.
Duly signed/stamped certification labels will be provided by the Accredited
Test Laboratory (ATL) to the Tissue Culture Production Facility at the time of issue
of certificate of quality of tissue culture raised plants for affixing on the packages
accompanying with dispatched planting material. The fee structure charged by ATL
will vary depending on the plant species, sample size etc.
6.5. Guidelines for Issuance of Certificate of Quality and Certification

Label:
“Certificate of Quality” and “Certification Labels” are to be issued if
samples of tissue culture raised plants have been tested for both freedom from
viruses and true to typeness / Genetic uniformity testing. However, genetic fidelity/
uniformity testing may not be required in some plant species. In such cases, only
“Certificate of Quality” may be issued without “Certification Label” clearly stating
that this certificate is only for Quality with respect to freedom for viruses. It may be
noted that tissue culture raised plants should be tested for all the known viruses
affecting the plant species being tested, as listed in the Standard Operating
Procedures (SOPs) for the purpose of issuance of certificate. The “Certificate of
Quality” should clearly mention the nature of testing conducted. Whenever
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“Certification Label” is issued both type of testing (i.e. virus indexing and genetic
fidelity/ uniformity testing) has to be conducted and samples are found virus free
and true to type/ genetically uniform.
6.6. Standards for Production of Tissue Culture Material:

Standards/guidelines for production of Tissue Culture material are currently
being prepared for different crops as per requirements, by DBT in consultation with
scientists/institutes, working in the area. The Accredited Test Laboratories will
issue the certificates only if Tissue Culture material is produced in conformity with
these notified guidelines. The Guidelines for potato have already been notified by
Ministry of Agriculture. Guidelines have been approved for other crops like
Banana, Sugarcane, Apple, Citrus, Vanilla and Black pepper.
6.7. Labeling of Tissue Culture Raised Plants/Propagules

Tissue culture plants/propagules shall be supplied in containers. A paper-
lined label of 12cm x 6 cm containing following information shall be affixed on the
container
1) Certified Tissue Culture Raised Quality Plants/Propagules
2) The label shall be rubber stamped with signature, name and designation
of the concerned Agency.
3) Colour of the label shall be diagonally yellow and opaline green.
4) The container should also have printed on it the kind, variety and name
of Institution.
5) Tissue culture raised plant producing Agency shall maintain the account
of labels printed and issued.
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CHAPTER 7:
Banana- Tissue Culture – (BTC) - Standards
The General Seed Certification Standards are basic and, together with the
following specific standards constitute the standards for approval of BTC. As the
name implies, these standards are applicable to tissue culture multiplied under
laboratory and greenhouse conditions as laid here. The General Standards are
amplified as follows to apply specifically to the BTC.
7.1. Eligibility requirements for BTC production:

 All micropropagation and greenhouse facilities must be approved as per
standards/ guidelines set by the competent authority. These must have a
changing area between double doors.
 Laboratory and greenhouse facilities used for production of plantlets shall be
maintained free of pests or vectors of banana pathogens. Failure to keep such
pests under control may cause rejection of all lots maintained in the facility. All
potting or growth media shall be sterile. Water sources used in the laboratory
or greenhouse operation shall be treated or otherwise rendered free of all
possible pathogens by the applicant.
 Hygienic conditions shall be strictly observed during micropropagation, potting,
planting, irrigating, movement and use of equipment and other laboratory and
greenhouse practices to guard against the spread of diseases or pests in the
facilities used for banana plant multiplication.
The greenhouse (protected environment) must be “insect proof” and be
equipped with a double-door entrance, provision for footwear disinfection prior
to entering the protected environment and insect proof ventilation screening on
intakes and exhaust openings. The persons entering the protected
environment should use Wellington boots (plastic boots) and change lab-coat
in the changing area to reduce the chances of inadvertent introduction of
vector insects clinging to clothes.
 The material being initiated must be of a notified variety and confirmed identity.
It must be duly documented with respect to origin.
 All samples of banana varieties being initiated should be tested in an
accredited laboratory and be free of viruses (Banana Bunchy Top Virus,
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Cucumber Mosaic Virus, Banana Bract Mosaic Virus, Banana Streak
Virus) and other endophytic or epiphytic bacteria and fungi.
 The basic material for sub-multiplication need to be obtained a fresh from the
nodal organization as soon as the maximum permitted number of passages
(as confirmed by DNA fingerprinting) of shoot multiplication with old cultures
has been completed.
 On application for inspection, the mother cultures as developed above are
eligible for certification. The micro propagation facility to be inspected must
have been approved by the competent Authority. All stocks must have a valid
variety identification and disease testing report at any time during multiplication
process.
In vitro multiplication of an imported variety or a non-notified variety can
be taken up by the industry exclusively for export purposes. Such varieties,
however, should be introduced following the approved guidelines of
Government of India.
7.2. Source of Seed:

 The facility should use recognized aseptic initiation and propagation
procedures (i.e. follow procedures and use equipment, which will maintain
sterile conditions as per standard tissue culture norms).
 The initiating facility must maintain following information on each variety for
review and audit by the competent authority at least once in a year: variety
identification, date of initiation, origin and testing results from accredited
laboratory.
 Tests must be carried out on a minimum of 0.1% (at least ten) plantlets for
each variety by an accredited laboratory. Such tests will be valid so long as
cultures of that particular batch are under production (subject to a maximum of
8 passages). No plant should contain (Banana Bunchy Top Virus, Cucumber
Mosaic Virus, Banana Bract Mosaic Virus, Banana Streak Virus) and other
endophytic or epiphytic bacteria and fungi.
 Valid pathogen testing results are required at the 2nd/3rd subculture stage
prior to the bulking up of the cultures.
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7.3.. Procedures and standard parameters for production of tissue culture
Banana plantlets
7.4. Minimum Quality Standards for growing of plants inside greenhouse:

gr
The following requirements must be met for production of plantlets:
 Effective sanitation practices including insect and disease monitoring and

prevention must be adhered to.
 No field-produced
produced banana plants can be grown in the protected environment
(greenhouse/polyhouse) along with tissue cultured plants.
 Varieties must bee separated by physical barriers (such as proper tagging),
which will prevent varietal mixture.

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 Before dispatch to the farmers, the tissue-cultured plants growing in the
nursery should be tested for the absence of the viruses (Banana Bunchy Top
Virus, Cucumber Mosaic Virus, Banana Bract Mosaic Virus, and Banana
Streak Virus) and clonal uniformity. For establishing clonal fidelity, the sample
size should be 0.1% of the batch size with a minimum of 10 plants.
 If testing performed by an accredited laboratory reveals the presence of
banned viruses, fungus or bacteria the tissue-cultured plants should not be
dispatched from the premises of the production lab and the entire material
should be destroyed.
 The concerned laboratory/agency producing the tissue culture raised material
should issue a certificate to the effect that BTC have been produced as per
guidelines
 The agency producing BTC will follow the labeling procedures
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CHAPTER 8:
Sugarcane- Tissue Culture – (STC) – Standards
following specific standards constitute the standards for approval of Sugarcane-
Tissue Culture (STC). As the name implies, these standards are applicable to tissue
culture multiplied under laboratory and greenhouse conditions as laid here. The
General Standards are amplified as follows to apply specifically to the STC.
8.1. Eligibility requirements for STC production:

 All micro propagation and greenhouse facilities must be approved as per
maintained free of pests or vectors of banana pathogens. Failure to keep such
 Hygienic conditions shall be strictly observed during micro propagation,
potting, planting, irrigating, movement and use of equipment and other
laboratory and greenhouse practices to guard against the spread of diseases
or pests in the facilities used for banana plant multiplication.
 The greenhouse (protected environment) must be “insect proof” and be
 All samples of sugarcane varieties being initiated should be tested in an
accredited laboratory and should be free of viruses (Sugarcane mosaic
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virus, Badnavirus , Yellow leaf and Luteovirus) and other endophytic or
epiphytic bacteria and fungi.
 The basic material for sub-multiplication need to be obtained a fresh from the
nodal organization as soon as the maximum permitted number of passages
(as confirmed by DNA fingerprinting) of shoot multiplication with old cultures
has been completed.
eligible for certification. The micro propagation facility to be inspected must
have been approved by the competent Authority. All stocks must have a
valid variety identification and disease testing report at any time during
multiplication process.
In vitro multiplication of an imported variety or a non-notified variety can be
taken up by the industry exclusively for export purposes. Such varieties, however,
should be introduced following the approved guidelines of Government of India.

review and audit by the competent authority at least once in a year:
variety identification, date of initiation, origin and testing results from
accredited laboratory.
 Tests must be carried out on a minimum of 0.1% (minimum ten) plantlets
for each variety by an accredited laboratory. Such tests will be valid as long
as cultures of that particular batch are under production. No plant should
contain (Sugarcane mosaic virus, Badnavirus, yellow leaf and Luteovirus) and
other endophytic or epiphytic bacteria and fungi (Testing for Redrot, Smut and
grassy shoot should also be included)
 Valid pathogen testing results are required at the 2nd/3rd subculture stage
prior to the bulking up of the cultures.
8.3. Minimum Quality Standards for growing of plants inside

greenhouses/ polyhouses:
The following requirements must be met for production of plantlets:
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 No field-produced sugarcane plants can be grown in the protected
environment (greenhouse/polyhouse) along with tissue cultured plants.
 Varieties must be separated by physical barriers (such as proper tagging),
which will prevent varietal mixture.
 Before dispatch to the farmers, the tissue–cultured plants growing in the
nursery should be tested for the absence of the viruses (Sugarcane mosaic
virus, Badna virus, yellow leaf and Luteovirus) and clonal uniformity. For
establishing clonal fidelity, the sample size should be 0.1% of the batch size
with a minimum of 10 plants per batch. Genetic variation up to 0.01% of the
representative
should issue a certificate to the effect that STC have been produced as per
guidelines
Since tissue culture is developing as a profitable industry with time,
certification of tissue culture plants (TCP) has been assuming more importance to
avoid any sort of variation or contamination. Therefore standard guidelines and
protocols for TCP production is becoming more stringent aimed at greater perfection
of the protocols.
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8.4. Procedures and standard parameters for production of tissue culture
Sugarcane plantlets
N.B.: Plants should be of superior quality in terms of growth, disease / pest resistance, drought
tolerance, high yield (fresh weight), sugar content etc. The explant should be healthy and free from
microbial infections, smut and, grassy shoot.
One set of mother plants must be maintained in the insect proof glass house as reference sample.
**Since sugarcane tissue culture is frequently confronted with endogenous bacterial contamination and
phenolic exudation, these should be eliminated using appropriate method.
***In sugarcane the number of passages can be up to 7 for subculture of shoots.
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CHAPTER 9:
Bamboo- Tissue Culture – (BaTC) - Standards
following specific standards constitute the standards for approval of BaTC. As the
name implies, these standards are applicable to tissue culture multiplied under
laboratory and greenhouse conditions as laid here. The General Standards are
amplified as follows to apply specifically to the BaTC.
9.1. Eligibility requirements for Bamboo- Tissue Culture production:

 All micropropagation and greenhouse facilities must be approved as per
maintained free of pests or vectors of bamboo pathogens. Failure to keep such
 Hygienic conditions shall be strictly observed during micropropagation, potting,
planting, irrigating, movement and use of equipment and other laboratory and
greenhouse practices to guard against the spread of diseases or pests in the
facilities used for banana plant multiplication.
 The greenhouse (protected environment) must be “insect proof” and be
vector insects clinging to clothes
 All samples of bamboo varieties being initiated should be tested in an
accredited laboratory and be free of endophytic or epiphytic bacteria and fungi.
Page 74
 The basic material for sub-multiplication need to be obtained afresh from
the nodal organization as soon as the maximum permitted number of
passages (as confirmed by DNA fingerprinting) of shoot multiplication with old
cultures has been completed.
eligible for certification. The micropropagation facility to be inspected must
have been approved by the competent Authority. All stocks must have a valid
variety identification and disease testing report at any time during multiplication
process.
 In vitro multiplication of an imported variety or a non-notified variety can be
taken up by the industry exclusively for export purposes. Such varieties,
however, should be introduced following the approved guidelines of
Government of India.

review and audit by the competent authority at least once in a year: variety
laboratory.
 Tests must be carried out on a minimum of 0.1% (at least ten) plantlets for
each variety by an accredited laboratory. Such tests will be valid so long as
cultures of that particular batch are under production (subject to a maximum
of 15 passages). No plant should contain endophytic or epiphytic bacteria and
fungi. Generally, viral infection is not seen in bamboos. However, there are
reports that suggest infection by Bamboo Mosaic Virus (BaMV) at the nursery
stage in India.
 Valid pathogen testing results are required at the2nd/3rd subculture
stage prior to the bulking up of the cultures.
9.3. Minimum Quality Standards for growing of plants inside

greenhouses/polyhouses
The following requirements must be met for production of plantlets :
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 No field-produced bamboo plants can be grown in the protected
environment (greenhouse/polyhouse) along with tissue cultured plants.
 Varieties must be separated by physical barriers (such as proper
tagging), which will prevent varietal mixture.
 Before dispatch to the farmers, the tissue-cultured plants growing in the
nursery should be tested for clonal uniformity. For establishing clonal fidelity,
the sample size should be 0.1% of the batch size with a minimum of 10 plants.
should issue a certificate to the effect that BaTC have been produced as per
guidelines
 The agency producing BaTC will follow the labelling procedures.
9.4. Nursery Development

The plants to be used for field trials would be supplied either in poythene
bags or bare-rooted (to save the transportation cost). In both cases the plants should
be kept in the nursery till they have recovered from the transportation shock. While
the plants in polythene bags may be kept directly in the nursery, the bare-rooted
plants should be transferred to the polybags prior to their transfer to the nursery. The
plants should be kept in the nursery for a minimum period of 10 days. However, many
a times the plants supplied are not of planting height. In such a situation it
would be necessary to prolong the stay of plants in the nursery (till the plants are at
least 18 inches in height) before their transfer to the planting site in an open field.
9.5. Requirements of a bamboo nursery (holding area)

 The nursery site should be on level ground and well drained
 It should be as close as possible to the plantation site
 It should have all necessary facilities for irrigation of plants
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 The site should be protected from animals
9.6. Managing a bamboo nursery

In the nursery the plants of different species should be kept in separate beds
to avoid any mixing. If there are more then one genotype for each species, then it
would be desirable to keep the plants genotype-wise.
 The approximate size of the nursery bed could be (8-10 m x 1-1.5 m)

 As much as possible, the beds should be prepared where there is some
protection (shade of a tree/thatch) for the plants from direct sunlight
 The beds should be leveled so that there is no accumulation of water
 Each bed should be properly labeled so that there is no mixing of plants
 In the nursery the plants should be irrigated periodically, and care should be
taken that they remain free of diseases
 Only healthy plants of uniform size (approximately) should be used for field
trials, particularly experimental trials
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CHAPTER 10:
Potato–Tissue Culture-Raised Mini-tuber-(PTCMT) Standards

for Certification
10.1. Eligibility requirements for PTCMT production:

The PTCMT to be eligible for production shall be from a source meeting the
following standards for laboratory and greenhouse facilities:
 Laboratory and greenhouse facilities used for production of plantlets/

microtubers or minitubers shall be maintained free of potato pests or vectors of
potato pathogens. Failure to keep such pests under control may cause
rejection of all lots maintained in the facility. All potting or growth media shall
be sterile. Water to be used in a laboratory or greenhouse operation should be
free from impurities.
 Hygienic conditions shall be strictly observed during micro propagation,
potting, planting, irrigating, movement and use of equipment and other
laboratory and greenhouse practices to guard against the spread of diseases
or pests in the facilities used for seed multiplication.
 All micro propagation and greenhouse facilities must be approved, as per the
standard/guidelines. These facilities must have a changing area between the
double doors.
 The greenhouse (protected environment) must be "insect proof" and be
environment should use Wellington boots (Plastic boots) and change lab-coat
 The material being initiated for producing PTCMT must be of
Registered/Notified variety and confirmed identity. It must be duly documented
with respect to origin.
 The plants of a potato varieties being initiated for tissue culture should be
tested in an accredited laboratory for freedom from the following viruses: PVA,
PVS, PVM, PVY, PVX, PLRV, PALCV, PSTVd and endophytic or epiphytic
bacteria and fungi.
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 Tests must be carried on a minimum of ten plantlets of each variety selected at
random. For virus testing ELISA or an equivalent method should be used, for
viroid RT-PCR should be used, and for fungi and bacteria light microscopy and
culturing on media should be used.
10.2. Sources of seed:

sterile conditions as per standard tissue culture morns).
review and audit by the competent authority once in a year: variety
laboratory.
 Tests must be carried out on a minimum of ten plantlets, selected at random,
for each variety by an accredited laboratory. No plant should contain PVA,
PVS, PVM, PVY, PVX, PLRV, PALCV, PSTVd and other endophytic or
epiphytic bacteria and fungi.
 Valid pathogen testing results are required prior to the initiation of micro tubes
production cycle or planting of test tube plantlets in the greenhouse.
 PTCMT shall be produced and multiplied from approved source in vitro plants
or microtubers, as per the prescribed procedure.
 PTCMT may be used as breeder seed for further production certified classes
of seed as prescribed in the Indian Minimum Seed Certification Standards.
 Concerned laboratory should issue a certificate to the effect that the PTCMT
has been produced with the standards as prescribed under their supervision.
10.3. Greenhouse/Controlled Environment Requirements:

 All micropropagation and greenhouse facilities must meet the standards given
above under eligibility requirements.
 The soil used for PTCMT production should not be infested with pathogen and
pests of potato, particularly the following: · Wart (Synchytrium endobioticum
(Schilb.) Perc.) and/ or cyst forming nematodes; Brown rot (Pseudomonas
solanacearum ) or non-cyst forming nematodes within the previous three
years; Common scab (Streptomyces scabies (Thaxt.) Waks. & Henrici)
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10.4. Inspection of Greenhouse/Controlled Environment facility used for
production of PTCMT:
The grower must notify the Competent Authority of his production plans well
in advance of the planting. The crop must be grown from approved basic source in
vitro plants or micro tubers, which were produced, in an aseptic environment. A
minimum of three inspections shall be made.
 The first inspection shall be made 35 days and 45 days after planting for plains
and hills respectively to verify growing conditions, extent of disease infection
and off types and also to confirm isolation requirement of one meter between
different varieties as to avoid mechanical admixture;
 The second inspections shall be made at 60-65 days after planting to verify off
types, disease infection if any and pathogen testing, on a representative
sample, comprising of 1% of the plants with a minimum of 5 and a
maximum of 25 plants sampled for each variety.
 The third inspection shall be made immediately after haulms cutting/
destruction in order to verify that haulms have been cut /destroyed by the
prescribed date and proper manner.
10.5. Field Standards of PTCMT at greenhouse:

a. G en e ral re q u i re m en t s
 Isolation: Minimum 1 meter between the different varieties grown in
greenhouse so as to avoid mechanical admixture.
 All micropropagation and greenhouse facilities must be notified (approved) by
DAC, as per the standards given above under eligibility requirements.
b . S p eci f i c re q u i re m en t s
Factor Maximum permissible limits
* Off types 0.05%
**Plants showing symptoms of - Mild mosaic 0.05%
**Plants showing symptoms of - Severe mosaic, 0.05%
Plants infected by brown rot (syn. Bacterial wilt) nil
(Ralstonia solamacearum)
*Maximum permitted before dehaulming; ** Maximum permitted at final inspection
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c. Seed Standards for PTCMT
Factor Standards for PTCMT

Weight of mini tuber (minimum) 1.0gm
Germination/sprouting (minimum) 90%
Varietal Purity (minimum) 99%
Pure seed 98%
Virus 0.01%
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EXPERIMENT NO. 1
G en eral I n st r u ct i o n s f o r U si n g T i s su e C u l t u re an d M o l ecu l ar B i o l o g y
L ab o rat o r y
Precautions:
 Wear a laboratory coat (use a pair of gloves when necessary).
 Do not eat, drink or smoke inside the laboratory.
 Aseptic techniques should be followed regularly at all the time.
 Never mouth-pipette any solution (acids, phenols, peroxides, organic solvents,
chloroforms, marcuric chloride, culture of microbes etc.)
 All microbial cultures are handled and treated as potential biohazards and
dispose them of after autoclaving or treating with 10% Formalin for 30 min.
 Dispose of all the wastes properly. Radioactive wastes should be stored
separately. Do not contaminate the lab door handles with radioactive
chemicals. Remove the radioactive gloves before opening doors.
 No chatting, gossiping and loud discussions inside the laboratory as it affect
your concentration on the work and that of your colleagues as well.
 Do not store food materials and drinks in the refrigerator where other
chemicals and solvents are stored. (Consumption of such stored food items
may lead to cancer).
 When handling laboratory animals, use gloves and wash your hands with soap
after the experiment.
Preparation of reagents:
 Store chemicals/ solvents at appropriate temperatures as mentioned on the
label.
 Use the highest purity chemicals and double distilled water for preparation of
reagents and solutions. (Weight of the substance, brand, batch No of the
substance, type of water used, amount of alkali or acid used to dissolve should
be recorded then arid there).
 Label all reagents. The label should contain the name of the solution/reagent,
concentration (% or Molar), date of preparation of the reagent and initials of
the person who prepared. Unlabelled solutions/reagents should be discarded.
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 Store the reagents in appropriate bottles and at appropriate temperatures.
(Light sensitive reagents and solutions should be stored in brown/dark bottles).
 Keep record of the reagent preparation and experimental details correctly and
completely. Store all the data sheets supplied along with fine chemicals and
enzymes. Note down the date of expiry, Batch No. and Catalogue No for labile
substances.
Good Laboratory Practices

 Never do direct mouth pipetting of infectious or toxic fluids; use a pipetting
device.
 Plug pipettes with cotton.
 Do not blow infectious material out of pipettes.
 Do not prepare mixtures of infectious material by bubbling expiratory air
through the liquid with a pipette.
 Use an alcohol-moistened pledget around the stopper and needle when
removing a syringe and needle from a rubber stoppered vaccine bottle.
 Use only needle-locking hypodermic syringes. Avoid using syringes whenever
possible.
 Expel excess fluid and bubbles from a syringe vertically into cotton pledget
moistened with disinfectant, or into a small bottle of cotton.
 Before and after infecting an animal, swab the site of injection with a
disinfectant.
 Sterilize discarded pipettes and syringes in pan where they were first placed
after use.
 Before centrifuging, inspect tubes for cracks.
 Use centrifuge trunnion cups with screw caps or equivalents.
 Avoid decanting centrifuge tubes; if you must do so. afterwards wipe-off the
outer rim with a disinfectant. Avoid filling the tube to the point that the rim ever
becomes wet with culture. Fill 3/4 only.
 Never leave a discarded tray of infected material unattended.
 Wrap a lyophilized culture vial with disinfectant-wetted cotton before breaking.
Wear gloves.
 Sterilize all contaminated discarded material.
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 Periodically, clean out deep-freeze and dry-ice chests in which cultures are
stored to remove any broken ampoules or tubes. Use rubber gloves and
respiratory protection during the cleaning.
 Handle diagnostic serum specimens (carrying a risk of infectious hepatitis)
with rubber gloves.
 Develop the habit of keeping your hands away from your mouth, nose, eyes
and face.
 Avoid smoking, eating, and drinking in the laboratory.
 Make special precautionary arrangements for respiratory, oral, intranasal, and
intra tracheal inoculation of infectious material.
 In laboratories working with organisms causing infectious diseases, a
biohazard' warning sign should be posted on the laboratory doors and in the
work places. In laboratories where radioactive experiments are done,
radioactive sign should be posted on the work place(s). These signs should be
clearly visible to others.
 Give preference to operating room gowns that fasten at the back.
 Evaluate the extent to which the hands may become contaminated with some
agents and operations; forceps or rubber gloves are available.
Experiments and Record keeping:

 Make sure the laboratory and laboratory benches are always kept clean (any
spillover of cultures, corrosive solvents should be cleaned immediately and
appropriately).
 Clean the balance immediately after your weighing, for any spillovers.
 Make sure that the glasswares, plasticwares, media, etc., are ready for the
experiment (if needed sterilize them previous day itself).
 Make sure that you have studied and understood the principle and
methodology of each experiment. Discuss the experiment with your
guide/expert in advance. Keep appropriate controls. Copy the protocols of the
experiment in the record notebook. Attach print outs of instruction sheets and
supporting materials to the record note book.
 In multi-step experiments it is important and prudent to check each step before
proceeding to the next step.
 Label all plates, tubes, cultures etc., correctly before starting the experiment.
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 Make sure that the equipments are in working condition and available for the
experiment and whether it requires prior permission and reservation, in
advance.
 Make sure that you know the proper operations of the equipments and if not,
assistance is available.
 Standard graphs, experiments using radioactive compounds and enzyme
assays should be done in triplicates. For standard graphs, plot all the points
(do not take the average) and draw the line, which passes through maximum
number of points.
 Use a record notebook of permanent binding. Do not record the data on loose
papers. Number all pages before using the record notebook. Use permanent
ink in writing the record. Don't rely on memory and enter all data and
observations directly into the record book then and there. Avoid abbreviations,
code names and code numbers in recording the data. Start new experiment on
a new page. Every experiment should have date, title and objective(s). A-ll
calculations should be done step by step and recorded neatly. Do not
overwrite on mistakes.
 Photographs, printouts, chromatograms, etc. should be labeled, to indicate
what they are, supposed to represent anc. then tape them directly onto the
notebook as soon as the experiment is over. Do not take the record book
outside the laboratory. (Relationship between two variables in experiments can
be best represented using graphs rather than in Tables. All such graphs
should be titled, dated and fixed in the record book on the same day).
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EXPERIMENT NO. 2
I n st ru m en t at i o n f o r a T i ssu e cu l t u re L a b o rat o r y
Various instruments are required for a tissue culture laboratory for in vitro
culture of plant cell tissue and organ. For regeneration and maintenance of aseptic
culture media preparation as well as development of regeneration of plant tissue
specialized instrumentations are required. These are essential for establishment of
a tissue culture laboratory.
Following equipments and facilities are essential for optimum growth of plant
tissues for in vitro culture.
Autoclave:
Autoclave or steam sterilizer is required for sterilizing tissue culture media
mercuric chloride, water and all the instruments like forceps, scalpels, petridishes
and empty culture vassals for required for inoculation. Generally tissue culture
media is sterilized in 121oC temperature, 15 pound pressure for 15 minutes.
Laminar Air Flow:

This is an instrument or close chamber which has the facility for sterile air
flow, a working platform on which all the inoculation work is performed. The filter
sterile air can through on the working platform to minimize the infection, a UV light
is provided for creating germfree environment of the chamber and a white
fluorescent light is fitted in side the chamber to see properly the objects during
inoculation. To check the air pressure a manometer is also attached. Inside the
chamber a spirit lamp or gas burner or a glass bid sterilizer is also fitted to facilitate
the flaming of forceps and scalpels.
Electronic balance:
It is required for measurement of all the chemical ingredients required for
preparation of synthetic media. Weighing of all the macro and microelements for
plant growth, vitamins, amino acids and plant growth regulators require a micro
balance. The readability level of the balance generally one mg or one microgram.
pH meter:
pH meter is required for adjustment of tissue culture media at 5.7 – 5.8. So
that the media can be used for tissue culture. Generally the pH of the media is
adjusted with 0.1N HCl in case of alkaline pH of the media and 0.1N NaOH for
Page 86
acidic pH of the media before mixing the sugar and agar agar in the media and its
subsequent boiling for poring into the culture tube or conical flax or culture bottle.
Hot air oven:

Hot air oven is required for drying all the utensils and glass good required
for tissue culture.
Refrigerator:
It is required for keeping all the stock solutions for tissue culture media (MS
media) to prevent the degradation of chemicals as well as growing of
microorganisms. Growth hormones are sensitive for higher temperature so these
are kept inside the refrigerator.
Microoven / Hotplate / Heater:

Microoven, hotplate or heater is essential to boil the media before poring to
the tube incase of semisolid media. Then it is plagued and autoclaved for further
use in inoculation.
Double distillation unit:

To obtain the pure distilled water this unit is required. In tissue culture
media the water should be mineral free and heavy metal free so that the content of
different macro and microelements remain constant as per the composition of the
media. This instrument has a boiler and a condenser so that the water can be
boiled and condensed to get single distilled water and then the single distilled
water can be used in another unit as feeding water to have the double distilled
water. The initial feeding water must be demonized water so a deionizer must be
fitter with the tap water which then can be used for single distilled water unit.
Culture room:
A culture room is one of the main component of the tissue culture unit.
Culture room is required for keeping the inoculated media in a fixed temperature,
light and humidity. The temperature of the culture room should be maintained at
22±2oC which is the optimum temperature for plant growth during in vitro culture.
Culture racks are fitted to keep the culture vessels on the lighted shelves. The light
intensity of the culture rack is generally 3000 lux which can be obtained with PAR
(Photosynthetically Active Radiation) light. The relative humidity of the room should
be 65 to 70%. Generally this room does not have any windows and the door should
be doubled door to minimize contamination of culture room.
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Greenhouse experiment:
Climate controlled green house is generally made up of polycarbonate and
fitted with fan and pad systems or AC to control the temperature. The light intensity
is controlled by putting the different net shed in side the green house. Humidity
monitor is also fitted in the room. A temperature sensor is kept inside the room and
the room temperature and humidity can be fixed with the help of a control panel.
So that fluctuation of room temperature may be regulated by operating AC with
timer or fan pad connected with water tank. Green house is required to acclimatize
the tissue culture plants in soil rite or coco pit or sterile vermin composed. Over
head sprinklers are fitted for liquid fertilizer, pestiside, fungiside and water
application in a regulated manner.
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EXPERIMENT NO. 3
S t u d y o f l ab o r at o r y eq u i p m e n t s f o r G en et i c f i d e l i t y st u d y
Acquaintance with basic facilities to Molecular Biology Laboratory,
examination and observation of different equipments and instruments for molecular
biological works in different laboratory practical are highly essential.
Following equipments required for use in molecular biological experiments
Centrifuge:
This instrument is capable of operating at speed of 15,000 rpm and consist
of motor and rotor. The rotor is solid with holes to hold the centrifuge tube that
contain the samples. Rotors are of two type i) Fixed angle rotor and swing out
rotor. The sample capacity varies from 0.5 ml to 250 ml. It is made up of aluminum
or titanium. The tubes are located diametrically opposite to each other so that the
load is distributed evenly around the rotor axis. The speed, time, temperature and
rotor is generally have to adjust to separate different types of macromolecules.
PCR (Polymerase Chain Reaction) machine:

The PCR (Polymerase Chain Reaction) machine is a devise which can
manage to regulate different temperature regime for DNA amplification in vitro with
a template DNA, DNA polymerase enzymes, all the four bases of DNA and a
stabilizing buffer in a PCR tube. The PCR consist of three basic stapes: i)
Denaturation ii) Annealing ii) Extension. Each of these steps is repeated 30-40
times or cycles with a initial denaturation of DNA at 94 oC for 5 min. Then
denaturation at 94 oC for 60 sec - 30 sec., annealing at 37 oC to 40 oC for 1 min
and extension at 72 oC for 1 min. A final extension at 72oC for 7 min is require to
complete PCR reaction.
Micro balance:
Electrically operate high precession balance is require for weighing of
chemicals. The rediability of microbalance is varied from 1 mg to 1 microgram.
Microbalance have single top loading pan fighted inside a glass chamber. They
have auto zero facility.
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UV/VIS Trans-illuminator:
Visible web length transilluminator is uses for visualization of protein and
enzymes bands on SDS-PAGE or Polyaccrylamide Gel stained with Coomassie
Brilliant Blue, silver nitrate or different enzyme staining solution under visible light.
UV transilluminator is used to visualized DNA gel stained with Ethedium Bromide.
UV-VIS Spectrophotometer:
This machine is used to determine the optical density of a soluation in
visible or UV range of light. It measures from 180 to 1100 nm wavelength. The
optical density (O.D.) of the liquid present in 1 cubic centimeter path length is
measured in this machine based on the Beer Lambert’s Law which states that the
absorption of light related to the concentration of the absorbed solution in use. In
visible web length the O.D. of protein, phenol, sugars, polysaccharides, etc. can
be measured whereas DNA and RNA can be quantified in UV spectrum at 260 nm
and 280 nm respectively.
Vortex:
This machine is used for homogenizing or better mixing of reagent or any
other solution in different rpm.
Hot-Plate:
Hot plat is used to warm up or boil any solution in a fixed temperature.
Magnetic Stirrer:
Magnetic stirrer is used to mix any solute in a solution with the help of a
magnetic bid placed inside the solution which is rotate in a specific velocity with a
rotated magnate placed inside this machine.
DNA Fluorometer:
This equipment is used to quantify the DNA following colorimetric principle.
It measures the O.D. at UV light and the amount of DNA can be obtained with a
DNA specific dye like Hoechest that intercalate in the double helix of the DNA.
Page 90
Gel-Documentation System:
It consist of a transilluminator with visible or UV light sources in a cabinet.
On the top of the transilluminator a CCTV camera is fixed to capture image of
agarose Gel having DNA or polyacrylamide gel having protein or enzymes which is
again attached with a computer. These gel image can be edited or analyzed with
the help of specialized software for quantification and quality analysis of DNA, RNA
or protein bands on gel surface. The banding pattern of the gel also analysed with
the help of special software to create the phylogenetic tree.
Table Top –Micro Centrifuge:

Micro centrifuge is the small centrifuge devise where only the fixed angle
mini rotor is fixed. The capacity of the liquid in each tube is varied from 0.5 ml to
2.5 ml. The rpm varies from 5000 rpm to 20,000 rmp depending upon the model of
the centrifuge. It is used for separate out different components of solution in small
volumes. Some times it also used to spin down low volume mixtures like DNA and
loading dye in gel loading.
Horizontal or Vertical Electrophoresis unit:

Electrophoresis consist of two units i) Power Pack and ii) Electrophoresis
unit. The agarose gel is cast in a acrylamide gel tray for DNA or RNA separation
or a polyacrylamide gel is prepared in between two glass plates for protein
separation. The wells can be prepared with the help of comb which is used to
loading place for the working sample inside the gel. The power pack is used to
supply constant current or volt to resold the gel run in specific buffer. The power
pack is connected with cathode (-) and anode (+) side of the electrophoresis
apparatus. The current flow is always from cathode to anodic end. Horizontal unit
is used for DNA or RNA separation while vertical unit is used for separation of
protein or enzymes or large unit can be used for manual sequencing of DNA.
Environmental Shaker:
This is basically an incubator with temperature control devise fitted with
platform shaker. It is used for bacterial culture, liquid suspension culture etc.
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Ice Flaking Machine:
This machine is used for formation of ice flakes which are required for
different biochemical and molecular experiments.
Liquid Nitrogen Container:

This metal container is made specially to store liquid nitrogen at -196 oC.
Liquid nitrogen is used to grind the tissue for DNA isolation or to preserve any
material in ultra low temperature.
Lyophilizer:
This equipment helps in concentrating the samples in power form in very
low temperature (-46-60oC). The vacuum is created with a pump and cooling is
made with condenser so that the DNA , protein sample can be dried to powder.
Ultra low Deep Freezer:

This a low temperature deep freezer that maintain very low temperature (-
86oC). It is used to storage tissue or chemicals for long term.
pH meter:
The pH meter is used to measure the ionic strength of a solution. This
instrument is equipped with an electrode that measure pH ranged from 0-14. pH
electrode is made up of thin glass, porous membrane sealed at one end of a hard
glass tube containing 0.1M HCl.
Page 92
EXPERIMENT NO. 4
In vitro meristem culture of sugarcane (Saccharum officinarum)
Sugarcane is a vegetatively propagated crop and it suffers from slow rate
of multiplication. Besides, diseases like red rot are very often damage the crop in
field. So to eliminate chances of disease in field as well as to generate large
amount of planting material in a short period in vitro culture of sugarcane is
proffered. To establish a germ free seedling in vitro cleaning and surface
sterilization of sugarcane sets are required. Meristems are excised from the
surface sterilized sets and are inoculated in sterile MS culture media under aseptic
condition and kept in culture room for organogenesis.
M at eri al s req u i red :

(a) Sterile MS culture media in culture tube (b) Sterile forceps and scalpels
(c) Sterile Petridishes (d) Sterile glass beaker (e) Sterile empty bottle (f) sterilized
Cotton (g) Laminar Air Flow (h) Gas burner/ spirit lamp (i) Sterile 0.1% mercuric
chloride solution (j) Sterile distilled water (k) Rectified spirit (l) 70% ethanol (m)
Labels and glass markers (n) Match box (o) explants(shoot apices)
P ro ced u re:
 Collect actively growing shoots from 3-4 months old field grown healthy
plants. Remove outer sheaths by wiping the sheath with rectified spirit. Tops
with growing apices are cut approximately 7-8 cm long with few whorls of
leaves.
 Keep all culture tubes, forceps, scalpel, petridish, beaker, inside the laminar
flow chamber after wiping the table top with alcohol and cover the front
door.
 Switch on the UV light of the laminar flow for 15 min to sterilize the laminar
flow environment.
 Switch on the light and air of the Laminar Air flow and leave for 10 min.
 Clean both the hand with rectified sprit and take shoot pieces of sugarcane
in a sterile beaker. The tops are washed for five minutes to remove the wax
on leaf sheaths in the soap solution and later rinsed four to five times with
distilled water until the soap solution is completely washed out.
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 Thoroughly rinse the shoot pieces in 70 per cent ethyl alcohol for one
minute. Again rinse with sterile water 4-5 times till alcohol is completely
washed away.
 Deep the shoot apices in 0.1% mercuric chloride (HgCl2) solution for
approximately 8 minutes
 Decant excess amount of HgCl2 solution and wash the treated pieces three
times with sterile distilled water.
 Keep all the surface sterilized shoot pieces on a sterile petridish from
beaker inside the laminar air flow.
 Wipe outer surface of each culture tube with rectified spirit and keep them in
order for culture.
 Carefully pick up the explants with sterilized forceps and placed in a sterile
petri dish using a fine forceps and scalpel. Flame and cool the forceps every
time after use, remove the outer leaf sheaths one by one.
 Give three to four longitudinal slits superficially with the scalpel. By giving
superficial transverse cuts at the base, remove the leaf whorls carefully
without exerting pressure on the internal tissues.
 Repeat the process until the apical dome with two to three leaf primordia is
exposed (suitable size of 3.5-4.0 cm)
 Flame the forceps on a gas burner or sprit lamp on one hand and take out
the cotton plug of the culture tube on the other hand and flame the mouth of
culture tube with media.
 Cool down the forceps for a while and take the explant with the help of
forceps and place firmly on the media of the culture tube.
 Cap the mouth of the culture tube with cotton plug after slight warming the
rim of the tube and keep back the forcep in the alcohol jar.
 Repeat the process for inoculation of shoot pieces in a number of tubes.
 Keep the culture tubes on a culture tube stand after proper labeling.
 Keep the culture tube after inoculation in a culture room. Maintain 25±2 0C,
with 16 hours daylight and 8 hours night break.( (2000-3000 Lux).and
observe after seven days.
 Take out all the utensils used in the culture from laminar flow and clean the
table top with alcohol and switch off the laminar air flow.
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 Due to phenolic exudates, the culture gets discolored at the place of contact
of the explants which hinders the absorption of nutrients resulting in its
drying. Shaking the tubes gently without opening the caps changes the
position of the explants and avoids the problem.
 After one week, transfer the explants to fresh medium if browning occurs
further another transfer to fresh medium is carried out. Initially, the growth is
slow and it takes about 30 to 45 days for new shoots to appear.
 After two cycles of transfer of the shoots to the solid medium, the shoot
apices establish themselves and they turn green in colour.
 Transfer the mass or the shoot into a new culture flask containing
multiplication medium (15-20ml) for which different concentrations of
cytokinin can be tried.
 Incubate the flask in the culture room under light and repeat the step 2- 4
times every 2-3 weeks so that a mass of shoots is formed. As a general
observation, sugarcane shoots can multiply 2.5 – 3 times initially every 2-3
weeks.
 In the laminar flow, under sterile conditions, remove the cap from the culture
bottle and use the forceps to carefully remove the explant from the
multiplication medium.
 Place the multiplied shoot mass on a sterile petridish or on the sterile glass
plate. Using a sterile scalpel carefully remove or cut plantlets away from the
mass of shoots/clumps.
 Remove the undesirable portion of the explant and using sterile forceps
rinsed in 70% ethanol and flamed, carefully place plantlets into the rooting
medium. Note that lower side of the shoot remains in contact with the media
and shoot remains straight.
 Carefully cap the culture bottle, label them properly and return them to the
rack for 2 - 4 weeks under the same condition. During this time, the shoots
will continue to grow; however, most of the plant’s energy will be focused
into producing roots.
P recau t i o n s:
 Dry the hands properly after weeping with alcohol to avoid the burning.
 Ware apron, head gear, face mask, before inoculation to minimize
contamination.
Page 95
 Do not hold the leaving tissue with hot forcep. Cool forceps after flaming to
avoid damage of the leaving tissue.
 Ensure the switch off the UV light before inoculation.
 Take care during putting of cotton plug after completion of inoculation due to
hot surface of the mouth of culture tube.
 Ensure the capping of culture tube properly to avoid infection.
 Do not take out the hands from laminar flow or touch the hands on any non
sterile object while inoculating.
Page 96
EXPERIMENT NO. 5
In vitro root regeneration of sugarcane from micropropagated shoots
The regenerated shoots are transferred to rooting MS medium
supplemented with 2-3 mg/l NAA or IAA for root regeneration.
Materials required:
 Sterile MS culture media in culture tube
 Sterile forceps and scalpels
 Sterile Petridishes
 Sterile glass beaker
 Sterile empty bottle
 Cotton
 Laminar Flow
 Gas burner/ sprit lamp
 Sterile 0.1% mercuric chloride solution, ,Sterile distilled water,Rectified
spirit, 70% ethanol, Labels and glass markers, in vitro grown soot tip
culture tubes of sugar cane.
Procedure:
 Prepare MS medium supplemented with 2-3 mg/l NAA or IAA and sterilize
in autoclave in the previous day.
 Keep all culture tubes containing MS medium supplemented with 2mg/l
BAP, forceps, scalpel, petridish, beaker, in side the laminar flow after
weeping the table top with alcohol and cover the front door.
 Switch on the UV light of the laminar flow for 15 min to sterilize the laminar
flow environment.
 Switch on the light and air of the Laminar Air flow and leave for 10 min.
 Clean both the hand with rectified sprit and take beaker containing soot
meristem explants.
 Wipe outer surface of each culture tube with rectified spirit and keep them in
order for culture.
Page 97
 Flame the forceps on a gas burner or sprit lamp on one hand and take out
the cotton plug of the culture tube on the other hand and flame the mouth of
culture tube with media.
 Cool down the forceps for a while and take the meristem slices with the help
of forcep and place firmly on the meddle of the media of the culture tube.
 Take out sugar cane shoots on to a sterile petridish with the help of sterile
forcep after flaming.
 Flame the scalpel and separate the sugar cane shoots carefully in to small
clumps and transfer on to a new medium containing 2mg /l NAA or IAA.
 Cap the mouth of the culture tube with cotton plug after slight warming the
rim of the tube and keep back the forcep in the alcohol jar.
 Repeat the process for inoculation of shoot tips in a number of tubes.
 Keep the culture tube on culture tube stand after proper labeling.
 Keep the culture tube after inoculation in a culture room and observe after
15 days.
 Take out all the utensils used in the culture from laminar flow and clean the
table top with alcohol and switch off the laminar air flow.
Observation:
Count the number of root producing shoots per tube without infection and
with infection. Record the number of shoot with root formation in each tube.
Calculate the infection percentage of tubes and root formation from shoot of sugar
cane.
Conclusion:
Establishment of aseptic plant regeneration of sugar cane takes 2-3
months. Root regeneration can be observed under sterio-zoom microscope or
visually and photographed. The in vitro grown rooted shoots are ready for
hardening in green house. All the shoots looks green with roots. Sometime albino
shoots also produce roots and become green in culture room. The percentage of
rooting and infection of sugar cane is …..% and …..% respectively.
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Precautions:
To prevent the browning of the media due to polyphenols 0.5 to 2% ascorbic
acids can be added into the medium or repeated culture of the explants in 15 days
intervals.
Hardening
The plants produced in tissue culture are maintained under high humidity
(above 90%) and it is gradually reduced over a period of 6 to 8 weeks. Plants are
later transferred to larger container with a compost based mixture. Plants are
maintained under shade and once they have started producing and maintaining
newer leaves/roots, they are the ready to be transfered in open nursery.
Page 99
EXPERIMENT NO. 6:
Meristem culture of ornamental plants in MS media.
P ri n ci p l e:
The vegetative propagated ornamental plants (viz. rose, gerbera etc) are
hosts to a number of bacteria, virus and many other pathogens. The bacterial and
fungal diseases can be controlled by the use of chemicals or improved agronomic
practices. However they can’ not control viral diseases.
With the advent of technique for culturing meristem – tips, developing virus
free plants from virus infected plants became rather easy. The technique is based
on the fact that the concentration of virus particles is much less in the rapidly
growing areas of shoot and root tips than in other parts of the plant. The plants
raised from meristems (0.3-0.5 mm long) on nutrient culture medium are generally
free from viruses. However viruses vary in case with which they can be eradicated.
Thermo therapy (35-40oC ) and chemotherapy (98- Azaguanine, 5-Fluorouracil,
etc.) are known to inhibit the replication of many viruses. Therefore, chemotherapy
and thermotherapy of plants before meristem tip culture or of meristem-tip during
culture have been successful in eliminating a number of viruses those are difficult
to eliminate by meristem-tip culture alone. Meristem-tip culture has become an
important tool for freeing banana, sugarcane and potato cultivars of virus infections
and increasing productivity. Dissect out meristem tips in this experiment from
apical shoots of mother plants grown for 5 weeks at 37 oC .
Apparatus required
 Culture bottle containing filter paper bridges and MS basal liquid Medium
supplemented with 2mg/l BAP, 0.1 mg/l GA3 2mg/l IBA and 20 g/l sucrose
 Glass Petri dishes containing whatman filter paper
 Conical Flask containing Sterile distilled water
 Forceps
 Needles
 Culture bottle containing 10 ml of 0.8% agar MS basal medium
supplemented with 2mg/l calcium pantothenate, 0.1 mg/l GA 3, 0.01 mg/l
NAA and 30g/l sucrose for potato.
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 Non-sterile items
 25% v/v Sodium hypochlorite solution, 4% w/v mercuric chloride, 95% and
70% ethanol, Tween 20, Laboline etc.
 Test tube stand
 Incubator at 37o C,
 Dissecting Microscope.
 Laminar air flow hood with Bunsen burner.
P ro ced u re:
I n d u ct i o n o f sh o o t s
 Cut out 10 mm long apical tips from the potted mother plants, remove
opened leaves from the apical tips and wash them under running tap water.
 Transfer the shoots to petridish containing whatman filter paper for surgical
removal of the shoot apex.
 Remove the rudimentary leaves with the help of sterile scalpel and needle.
Cut 0.3-1 mm long meristem tip.
 Open culture tube under flame and transfer the meristem tip to the
depression in the filter paper bridge with the help of scalpel tip.
 Flame the mouth culture tube and close it with plug. Incubate the culture
tube at 25oC under 16 hour photoperiod (300mm one fluorescent tube of 20
watt.). Within 5-6 weeks a dense cluster of proliferating shoots develops.
 After about two weeks further growth, these shoots are excised and
inoculated on agar gelled medium in fresh culture tubes under aseptic
conditions. These culture tubes are incubated at 25 0C under 16 hour
photoperiod. In 3-4 days root start developing and within 20 days 60-90 mm
long shoots develop. The plantlets are ready for multiplication.
M u l t i p l i cat i o n
The in vitro plantlets obtained from meristem tips are multiplied in culture
bottles using nodal cutting. The in vitro plantlets are tested for virus by ELISA. The
virus free plantlets are further multiplied either for in vitro conservation or for speed
production of germplasm.
Page 101
EXPERIMENT NO. 7
Suspension culture technique for production of secondary metabolites
A cell suspension culture consists of cell aggregate dispersed and growing
in agitated/ moving liquid medium. It is normally initiated by transferring a piece of
undifferentiated and friable calli to liquid medium, which is continuously agitated by
a shaker. This technique is widely used for production of secondary metabolites.
Materials required:
(a) Agar gelled MS + 1-2 mg/l 2,4-D; Liquid MS + 1-2 mg/l 2,4-D; (b) Sterile
forceps and scalpels (c) Sterile Petridishes (d) Sterile glass beaker (e) Sterile
empty bottle (f) Cotton (g) Laminar Flow (h)Gas burner/ sprit lamp (i) Sterile 0.1%
mercuric chloride solution (j) Sterile distilled water (k) Rectified spirit (l) 70%
ethanol (m) Labels and glass markers (n) Match box (o) germinated seedlings with
cotyledon
Procedure
 Collect the aseptically germinated seedlings when the cotyledons are fully
expanded and the epicotyl is beginning to emerge. Usually this will occur
when the seedlings are 2-week old.
 Place each seedling on a sterile slide/ petri dish and prepare the explant.
 Various explants can be used. Excise the shoot apex with and without
cotyledons and insert the stem base into the medium, cotyledons, and
hypocotyl section from the decapitated seedling, etc. Place the explants on
the following medium: MS + 1-2 mg/l 2,4-D
 Incubate the cultures in dark at 25°C. Callus will be produced in 3-4 weeks.
 Cut small pieces of callus of -0.5 g fresh weight and subculture on the same
fresh medium for proliferation.
 Prepare MS liquid medium without agar. For experiment purposes 15 ml
medium in a 125 ml Erlenmeyer flask is put. Sterilize the opening of a flask
with the flame of burner in the hood.
 Transfer a piece of callus to a sterile petridish. Gently break the callus of -2
cm diameter with forceps into 20-30 small pieces.
 Transfer the small pieces of callus to the liquid media using forceps.
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 Heat sterilize the opening of a flask and place the sterile cap on the flask.
Prepare replicated samples.
 Incubate on a gyratory shaker set at 125 rpm which should be placed in a
temperature controlled room.
 Subculture every week. For the first few subcultures,- remove a portion of
the spent medium and replace with fresh medium with the help of a large
bore sterilized pipette. When the cell mass has about doubled, carefully split
the culture into two flasks with an equal volume of fresh medium. Repeat
the incubation cycle.
 Once the suspension culture becomes established and consists of finely
dispersed cell clusters and aggregates, a dilution ratio of 1.: 4 to 1 : 10 old
culture to fresh medium should be possible on a 7-10-day basis to maintain
the cell line. Cell suspension can be seen under a microscope by taking an
aliquot from the flask on a glass slide under sterile conditions.
 Secondary metabolites were harvested from the suspension culture through
centrifugation
Page 103
EXPERIMENT NO. 8
Synthetic seed formation from somatic embryos
A typical synthetic seed is a aseptically encapsulated bids of sodium
alginate of somatic embryo or shoot buds for preservation and growth. It following
parts such as: (a) Plant propagule like somatic embryo or shoot buds (b) Matrix,
is a gelling material encapsulating plant propagules which incorporate nutrients,
antibiotics or other essential additives. (c) Seed shell these are the artificial seed
coats prepared with complex mixture of alginate-gelatin which was used to develop
the coat system for encapsulation.
Alginate as a straight chain, hydrophilic colloidal polyuronic acid composd of
hydro-β-D Mannuronic acid residues with 1-4 linkages. The major principle
involved in the alginate encapsulation process is that sodium alginate droplets
containing the somatic embryos when dropped into the CaCl2.2H2O solution form
round and firm bead due to ion-exchange between Na + in sodium alginate with Ca+
in CaCl2.2H2O solution. The hardness or the rigidity of the capsule depends upon
the number of sodium ions exchanged with calcium ions. Hence the concentration
of the two gelling agents, i.e. sodium alginate and CaCl2.2H2O and the complexing
time should be optimized for the formation of the capsule with optimum bead
hardness and rigidity. In general 2-3% sodium alginate upon complexing with 50-
75 mM CaCl2.2H2O for half-an-hour gives optimum bead hardness and rigidity for
the production of viable seeds. Since somatic embryos lack seed coat and
endosperm that provide protection and nutrition for the zygotic embryos in the
developing seeds. Therefore, addition of nutrients and growth regulators to the
encapsulating matrix is desired as these supplements enhance efficiency of
germination and viability of encapsulated somatic embryos.
Material Required:
(1)Sterile MS medium (2) Explants - Somatic embryos of rice (3) Sodium
alginate (4)Calcium chloride (5) Sterile petridish (6) Sterile glass beaker (7) Sterile
pasture pipette/ spatula (8) Sterile plastic cryo tubes (9) Laminar Flow (10) Spirit
lamp/ gas burner (11) Rectified sprit (12)Labeles and glass markers (13) Match
box (14) Cottons (15) Sterile filter paper (16) Sterile distilled water
Page 104
Procedure:
 Rice embryos/ axillary buds/ shoot tips are carefully isolated from aseptic in
vitro cultures and blot dried on sterilized filter paper.
 The gelling mixture, consisting of half strength of liquid Murashige and
Skoog. (1962) medium, 2% sodium alginate, 3% sucrose and 50 mM
calcium chloride solution (CaCl2), was autoclaved at 120oC for 20 minute
under 15 lb pressure in the previous day.
 The propagules are then mixed in sterile 2% sodium alginate prepared in
nutrient (MS) medium with 3% sucrose.
 The propagules are then picked up manually by forceps or sucked with the
help of dropper and dropped into a solution of 50 mM calcium chloride in a
sterile beaker for about 30 minutes.
 After the incubation period, the beads (synthetic seeds) are recovered by
decanting the calcium chloride solution and washing them in sterile water
for 3 to 4 times before storage.
 Finally the synthetic seeds are stored at a temp of 10 0C in cryo tubes.
 The moist condition was maintained by spraying encapsulated embryos/
shoots with sterile distilled water at regular day of interval.
 Synthetic seeds were also cultured in MS medium without any growth
hormone to tested the viability of the synthetic seed after weeks intervals.
Precautions
 Use aseptic condition all the time during seed formation.
 Full encapsulation with Sodium Alginate of explants is essential to prevent
infection.
 Use of low amount of antibiotics in the MS medium could preserve well the
explant.
 Proper time should be given for gelling the synthetic seeds in Calcium
chloride.
 Keeping of seeds in low temperature prevents reduction of viability.
 During freezing maintain moister of the synthetic seeds.
 Do not dip sodium alginate containing pipette or spatula in calcium chloride
solution to avoid the immediate gel formation and blockage of pipette.
Page 105
EXPERIMENT NO. 9
Extraction of genomic DNA from leaf samples by CTAB method.
C-TAB (Cetryl Trimethyl Ammonium Bromide) method of DNA isolation was
developed by Marry and Thompson in 1989 as an alternative method against
lengthy and expensive, low yield Cesium chloride – ethidium bromide – ultra
centrifugation procedure. That method required isolation of nuclei and the product
was only degraded DNA. The specific characteristics of plant like presence of rigid
polysaccharides cell wall, pigments, chemicals, secondary of metabolites in cell
cytoplasm found in various plant species necessitate C-TAB method of DNA
isolation and purification.
Principle:
The DNA can be isolated by using the cells in presence of buffer containing
C-TAB in presence of EDTA followed by further extraction with phenol and
chloroform mixture to remove the proteins and other constituents of the tissue.
Addition of absolute ethanol removes the polysaccharides and secondary
metabolites which form the major constituents of the plant cell. β-mercaptoethanol
is cationic detergent and also acts as an antioxidant and prevent the oxidation of
nucleic acids as well as helps in the removal of lipid by emulsification. CTAB is
positively . C-TAB is +ve charge particles which form complex with –ve charge
DNA and precipitate DNA. NaCl helps the break anionic bonds between
biomolecules. EDTA used as chilating agent which deactivate the coenzymes and
removing Ca+ and Mg++.
Materials required:
(1) Leaf samples, (2) Temperature controlled water bath (3) Mortar and
pastel (4) Refrigerated centrifuge (5) Sterilized centrifuge tubes (6)Steriliged
Eppendorf tubes (7) Parafilm (8) Tissue paper (9) Spectrophotometer (10) Sterile
glass hooks
Page 106
Reagents:
DNA extraction buffer (100 ml)
Sl Final concentration Stock solution Quantity
No. strength taken
1 100 mM Tris 1M 10 ml
2 20 mM EDTA 0.5 M 4 ml
3 1.4 M NaCl 3M 46.66 ml
4 2% CTAB - 2g
5 0.2% β-marcaptoethanol - 200 µl
6 Sterilized double distilled water - 41.14 ml
Total 100 ml
24:1 Chloroform : Isoamyl alcohl

This solution is prepared by mixing 96 ml of chloroform (A.R. grade) + 4 ml
of isoamyl alcohol for 100 ml of solution. This mixture is prepared immediately
before use.
70% ethanol (100 ml)

70 ml of dehydrated alcohol (100%) is make upto 100 ml with sterile double
distilled water.
T10E1 buffer (100 ml)

Final concentration Stock strength Quantity required
10 mM Tris 1M 1 ml
1 mM EDTA 0.5 M 200 µl
Double distilled water - 98.8 ml
Liquid nitrogen
Obtained from commercially and stored in liquid nitrogen container.
Isopropanol
Isopropanol of AR grade was kept in -20 oC or chilled tray of the refrigerator
to bring down its temperature at chilling stage.
Procedure:
 2 gm of leaf samples of aseptically grown Vigna radiate was taken in to a
sterile mortar pastel and grind with liquid nitrogen to make a fine powder.
 4 ml of DNA extraction buffer is warmed at 60oC in a temperature controlled
water bath inside a sterile centrifuge tube.
Page 107
 Once the extraction solution is reached at 60oC, the grind powder of leaves
were put inside the buffer and caped it tightly. The tissue and extraction
buffer ration was in 1:2 ratio. The centrifuge tube containing leaf sample
and extraction buffer was incubated at 60oC for 1 hour. Two to three times
occasional mixing was made with a sterile glass rod to break the clumps of
the material inside the tube and through mixing.
 Samples were taken out from water bath and keep in room temperature to
teach back the sample at root temperature. Then an equal amount of i.e. 4
ml of 24:1 chroloform:isoamyl alcohol was added to the sample and shaken
gently for 10 to 15 min.
 After a through mixing of samples and chloroform:isoamyl alcohol like a
emulsion, it was centrifuged at 10,000rpm for 20 mins at 10 oC in a
refrigerated centrifuge.
 The supernatant was transferred to a sterile centrifuge tube and repeat the
step 5 once more.
 The supernatant was transferred to another sterile centrifuge tube with the
help of micropipette and about 0.6 volume of chilled isopropanol (2.4 ml)
was added to it. The solution was mixed gently and after sometime the DNA
will precipitated or floated like a threads.
 DNA was pulled out from the solution with a sterile glass hook and rinsed
with 70% ethanol to dry it properly and transferred into a sterile eppendorf
tube. If the there is no any thread formation, keep the solution at -20 oC for
over night and the precipitation will formed. Then next day the solution can
be centrifuged at 10,000 RPM for 10 mins at 10 oC and the pellet can be
rinsed with 70% ethanol twice.
 The mouth of the eppendorf tube was sealed with a parafilm and perforated
and the DNA was died under a lyophilizer.
 The lyophilized DNA powder was dissolved with 100 -200 µl of TE buffer
and stored at 4 oC for further use.
Precautions:
 Always collect tender leaf tissue from meristematic region because the cell
density is higher in it. The old and mature tissue has rigid cell wall which is
not easy to break.
 The glossy leaf with waxy coating can be wiped with ethanol followed by
diethyl ether to remove the germs and waxy layer of the tissue.
Page 108
 Mortar pastle should be kept at -20oC before use. All the plastic ware and
solutions should be sterilized before use and sterile gloves should be used
during handling of DNA and extraction.
 Chloroform : Isoamyl alcohol should not be added immediately after
removal of sample from 60oC water bath. After incubation at 60oC for 1
hour the solution should be bring down to room temperature and then
chloroform : isoamyl alcohol should be added.
Page 109
EXPERIMENT NO. 10
Purification of genomic DNA
Pure DNA free form protein contamination will have an A260/A280 ratio close
to 1.8. If phenol or protein contamination is present in DNA preparation, the A 260 /
A 280 ratio will be less than 1.8. If RNA contamination is present in the DNA, then
A260/A280 ratio may be grate than 1.8. Pure RNA preparation will be having an A
260/A280 ratio close to 2.0. Before going for purification of DNA its quantification
should be done so as to know the nature of contamination i.e. protein or RNA
present in the sample.
P ri n ci p l e:
The RNA contamination from DNA preparation is removed by treating the
crude DNa with RNase A and proteins are removed by treating with proteinase K.
Extraction of crude DNA with phenols: Chlorophorm: Isoamyl alcohol (25:24:1) also
helps to remove the proteins from DNA preparations.
Materials required:
Microcentrifuge, DNA from Vigna radiate leaf, Rnase A, TE buffer,
Eppendorf tubes, Chloroform, Isoamyl alcohol, phenol, ethanol,
spectrophotometer, lyophilizer
Procedure:
 10g of RNase A was dissolved in 1 ml of 10 mM Tris HCl (ph 8.0) and 15
mm NaCl n a 1 ml microcentrifuge tube.
 The tubes were treated in boiling water bath at 100 oC for 15 min to denature
contaminating DNase and then cooled to room temperature.
 RNase at 50 µg/ml at crude DNA solution was added and incubated at 37 oC
for 1 h.
 Then equal volume of phenol (TE saturated) : chloroform: isoamyl alcohol
(24:1) in 1:1 ration was added and mixed thoroughly.
 Samples were centrifuged at 10,000 rpm for 5 min at room temperature and
the supernatant was transferred to the new eppendorf tube.
 3m sodium acetate (pH 4.8) was added in 0.1 volume to the crude DNA
sample volume and mixed thoroughly.
Page 110
 2.5 times chilled ethanol was added and mixed with the sample gently to
precipitate the pure DNA.
 DNA was precipitated and the clumps of DNA can be hooked with a sterile
loop or centrifuged at 10,000 rpm.
 The DNA clumps or ppt of DNA was rinsed with 70% ethanol and dried
under vacuum.
 The dried DNA samples was dissolved in TE buffer and kept for O.D.
checking and Gel electrophoresis for further PCR reaction.
 1 ml of TE buffer was taken in a quartz cuvette and 5 micro litter of DNA
was added to it .
 O.D. was recorded at 260 nm and 280 nm and the concentration of DNA
was calculated with the formula as follows:
O.D. at 260 x 50 x dilution factor /1000 = (ng/µl DNA)
Precautions:
 Shaking of DNA vigorously in phenol solution may damage DNA.
 While taking O.D. the volume of the TE buffer should be exactly 1 ml or else
the path length of spectrophotometer will be empty and the O.D. reading
error will be there.
Page 111
EXPERIMENT NO. 11
Study of quality checking of DNA by agarose Gel electrophoresis
Electrophoresis is a technique used to separate and purify
macromolecules especially proteins and nucleic acids that differ in size and
charge. When charged molecules are placed is an electric field, they migrate
towards either the positive or negative pole according to their charge in contrast to
proteins which can have either a net positive or net negative charge. Nucleic acids
have a constant negative charge imparted by their phosphate backbone so as to
migrate towards the anode (+) for the cathodic (-) end of a supplied current from a
power pack.
Principle:
By using gel with different concentrations, DNA fragments of different sizes
can be resolved. Higher concentration of agarose facilitate separation of small
DNA’s while low concentration agorase gel facilate separation of large DNA
molecules. In TAE and TBE buffer DNA fragments will migrate at different rates
due to differences in ionic strength. Buffers not only establish pH but also provide
ions to support conductivity. Ethidium bromide is fluorescent dye that intercalates
between bases of nucleic acids and allows very convenient detection of DNA
fragments in gel. Binding of ethidium bromide to DNA alters its mass and rigidity
and therefore its mobility.
Materials required:
(a)Horizontal electrophoresis apparatus and power pack (b) Gel casting tray
(the end of the tray should be closed with cello tape while casting the gel and then
removed prior to electrophoresis). (c) Comb (d) Micropipette (e) Sterilized
micropipette tips (f) Conical flask (g) Agorose (h) Microbalance (i) Electrophoresis
buffers
i) 10X Tris Borate EDTA buffer (TBE) pH 8.2 (100 ml)

0.9 M Tris HCl (11.3 g Tris HCl)
0.9 M Boric acid (5.5 g Boric acid)
0.025 M EDTA (0.93 g EDTA)
Page 112
ii) 10X Tris Acetate EDTA (TNE) buffer (pH 7.4) (100 ml)
10 mM Tris
1M NaCl
10mM EDTA
iii) 50 X TAE buffer pH 8.0 (100 ml)

100mM Tris acetate
1M NaCl
10mM EDTA
(use 1X in agoarose gel)
iii) Loading dye (10X) pH 7.6

TAE 1X
Bromophenol Blue 0.25%
Xylene Cyanol FF ) 0.25%
Glycerol 50%
iv) Ethidium bromide

10mg/ml double distilled water.
Procedure
 Agarose (0.8%) gel was prepared in 1X TAE buffer and casted in gel
casting tray by adding 10µl ethedium bromide.
 Then comb was inserted in the gel and it was allowed to solidify at room
temperature.
 After the gel is solidified the comm. was removed carefully and the cello
tape of both end of gel is removed.
 Gel was inserted in side a gel chamber of the horizontal gel electrophoresis
filled with TAE buffer.
 10µl of DNA sample along with 3 µl of loading dye was added to each
sample and mixed properly.
 The samples were spin down in microfuge with a short run at 6000 rpm.
 The samples were loaded in each well of the gel . A lane is also loaded with
known amount of lambda DNA sample.
 Gel was run at 55 V for 1 h connecting the electrodes at right end of the
power pack as well as gel.
Page 113
 Gel was visualized under UV transilluminator fighted in Gel Docomentation
system.
 Quantity of the DNA in the gel sample was estimated by its comparison of
O.D. and band volume with standard.
 DNA image is taken with Gel documentation system.
Precautions:
 Taking out of gel is tricky and it should be done carefully so that the gel well
should not be disturbed.
 Ethedium bromide is very hazardous and it should be handled wearing a
glove. Do not spill out the chemical here and there or on the working table.
 UV light is health hazardous and mutagenic so while visualizing the gel
under UV transilluminator a UV protected spec must be needed to protect
the eye from the UV radiation.
Page 114
EXPERIMENT NO. 12
Genetic Fidelity study of banana plantlets using ISSR primers
Polymerase Chain Reaction (PCR) is a technique used to selectively
amplify in vitro a specific segment of total genomic DNA a billion fold. The
essential requirement of PCR is the availability of short oligonucleotide (10 to 25
nucleotide base) called primers having sequences complementary to either end of
the target DNA segment (the template DNA) to be synthesizd in a large amount.
The PCR involves 3 basic steps which constitute a following cycles;
1. Denaturation of target DNA at 94oC.
2. Annealing of the primers to the template DNA at 40-55 oC.
3. Primer extension by addition of nucleotides to the 3’ end of the
o
primers at 72 C by Taq DNA polymerase.
As the number of PCR cycle increases the amount of target DNA
synthesized exponentially i.e. double in each cycle. Therefore one initial double
stranded DNA molecule there will be 2n molecules, where n=number of cycles.
This feature makes PCR very suitable procedure having the ability to detect very
low concentration of target sequences and amplify them to a level at which they
can detected by simple ethidium bromide staining. Availability of thermo-stable
DNA polymerase like Taq DNA polymerase from Thermus aquaticus has facilitated
the PCR reaction possible during denaturation of DNA at higher temperature when
the enzyme remain active without degradation.
Requirements:
Sterilized PCR tubes, microtips, micropipettes of different size, DNA
samples, 10X PCR buffer, dNTPs (dATP, dGTP, dCTP, dATP), Taq DNA
polymerase, Operon primers, DNAse free PCR water, PCR machine, 50X TAE
buffer (2M Tris-acetate, pH 8.0) +.0.05 M EDTA, ph 8.), 10X loading dye (0.25%
Bromophenol blue + 0.25% Xyline Cyanol FF + 50% Clycerol + 1X TAE), Ethidium
bromide (10mg/ml). Agarose, Horizontal gel electrophoresis unit with power pack,
Gel documentation system with UV transilluminator.
Page 115
Reaction mixture for PCR:
Ingredient Amount per reaction (for 25.00 µl)
PCR water 16.0 µl
10X PCR buffer 2.5 µl
dNTP mix 0.2 µl
Taq DNA polymerase 0.3 µl
Primer 5.0 µl
Template DNA (20 ng/µl) 1.0 µl
----------------------------------
Total 25 .0 µl
Procedure:
 The master mix was prepared except template DNA and primer and
distributed in PCR tube in ice box.
 Then template DNA and primers were added and PCR reaction mixture was
vortex thoroughly and centrifuged for while to settle down all the mixture
properly.
 The reaction was carried in a Parkin Elmar thermocycler for 45 cycles with
programming with following steps;
Cycle 1: Denaturation at 94 oC for 4 min
Primer annealing at 40 oC for 1 min
Primer extension at 72 oC for 2 min
Cycle 2-44: Denaturation at 92 oC for 1 min
Primer anneling at AT oC for 1 min
Primer extension at 72 oC for 2 min
Cycle 45:Final extension at 72 oC for 7 min Hold at 4oC for 10 min
 After the PCR is over the PCR products were kept in the deep freeze for
over night.
 Next day the amplified products were mixed with loadng dye and run into a
0.8 agarose gel under 55 V with a power pack using horizontal
electrophoresis and power pack for 3 h. The running buffer used for gel
casting and electrophoresis was 1X TAE buffer.
Page 116
 Once the loading dye is moved to the bottom of the gel the DNA gel was
removed from electrophoresis and stained with 0.5 µl/ml of gel for 15 min
and visualized under UV light in Gel Documentation system.
 Then the gel was photographed and analysed using Gel Doc system.
Result:
Clear bands of amplified products of different banana varieties were
observed on the gel. The results is presented with sequences of each primers
used, number of bands amplified, percentage of polymorphism. DNA photograph is
fixed in the record with a marker DNA ladder of 100bp to 3000 bp. The bands were
scored, similarity coefficient values were determined and a phylogenetic trees were
prepared applying NTSYS software. It was found that ………… varieties are
genetically more similar with ………. varieties.
Page 117
(To be used by only Recognized companies under NCS-TCP)
Annexure-1A
Intimation form for Virus Indexing of Plant Tissue/Stock Culture(s)
(The intimation should reach the Accredited Test Laboratory at least two weeks before the
sample(s) is (are) sent)
1. Name/ Address of the tissue culture
production facility:
2. Recognition details under NCS-TCP Registration No.
Certificate No.
Validity of Certificate:
3. Name of authorized person & contact

details (Telephone/ Fax/Mobile/E-Mail):
4. Details of plant sample to be tested: (Each sample should have at least 0.5gm
tissue/virus/test for all the known virus to be tested*)
5. Number of sample to be tested Plant species
6. Any additional information:
Date:
___________________
(Signature/Name of Applicant)
*List of viruses to be tested for plant species available at NCS-TCP website

(http://www.dbtncstcp.nic.in)
………….……………………… cut here ………………………………………….
ACKNOWLEDGEMENT
This is to acknowledge the receipt of “Intimation” for testing of plant tissue/ stock culture(s) from
the tissue culture production facility: (Name/Address)
………………………………………………………………………………………………………….. Dated
………………………….. We request you to kindly send the samples along with the “Application Form
for Virus Indexing of tissue stock culture(s) and testing fee of Rs: ……………........./- (in
word…………………………………………………….) through DD/cheque no. ……………………… in favour of
……………………………………………………………………for virus indexing for all the known viruses
Date:
Place: ---------------------------------------------------
Signature & Name of Director/HOD of
Accredited Test Laboratory
Note: Kindly enclose the self addressed and stamped envelope with intimation form.
Page 118
Annexure-1B
Intimation form for (Virus/ genetic fidelity) Testing for Batch Certification of Tissue
Culture Raised Plants
(To be submitted only by tissue culture production facility recognized under NCS-TCP.
The application should reach the ATL at least two weeks before the sample(s) is (are) sent)
1. Name and Address of the recognized tissue culture

production facility:
Certificate No.
3. Name of authorized person & contact details (Telephone/
Fax/Mobile/E-Mail):
4. Details of tissue culture plants required to be sampled: (Each sample should have at least
0.5gm tissue/virus/test for all the known virus to be tested* and an additional 1.0 gm for genetic
fidelity testing. Sample(s) from mother plant/stock culture is (are) also to be sent for genetic fidelity
testing)
Number of sample to be Plant species Batch number Batch size
tested.
5. Is the above batch derived from indexed stock cultures/mother plant: Yes/No
If yes!
5.1 Please indicate the sample registration number of stock culture from which the batch has
been derived in case the indexing has been done by ATL under NCSTCP:
5.2: If test of stock culture has not been done by ATL then give the details of indexing lab:

Date: _____________________
(Signature/Name of Applicant)
*List of viruses to be tested for plant species available at NCS-TCP website http://www.dbtncstcp.nic.in)
……………………………….……………………………….cut here…………………………………………………..
ACKNOWLEDGEMENT
This is to acknowledge the receipt of Intimation for testing and batch certification of tissue culture
raised plants from tissue culture production facility: (Name/ Address)
…………………………………………………………………………………………………………. We request you to
kindly send the samples along with the application for testing certification of tissue culture raised
plants and fee Rs…………………(in word…………………………………………………….) through DD/cheque
no. ……………………… in favour of ……………………………………………………………………
Date:
Place:
Signature/Name of Director, Institute

or HOD of Accredited Test Laboratory Note: Self addressed and stamped envelope to be enclosed
with the intimation form.
Signature and seal
Page 119
To be used by only Recognized companies under NCS-TCP)
Annexure-2 A
Application for Virus Indexing of Plant Tissue/Stock Culture (s)
1. Name/ Address of the tissue

culture production facility:
2. Recognition details under NCS- TCP: Registration No.

Certificate No.
3. Name of authorized person &
contact details(Telephone/ Fax/Mobile/Email):
4. Details of plant sample to be tested: (Each sample should have at least 0.5gm
tissue/virus/test for all the known virus to be tested*)
Sam Plant Vari *Nature of 20 digits Sample registration Number** (to be allotted by ATLs)
ple spec ety sample
No. ies
Leaf : 1 Date/Month/Year Nature M
Sample No.
Stem : 2 of sample of P
Rhizome: 3 TCPF Variety received sampl
Tuber : 4 es
(Registrat Specie 1
Leaf: 1
ion no.) s ATL Stem
(Regist :2 S
D M YY
ration Rhizo C
no.) D M me : 3
Tuber 2
:4
5. Sample drawn by: Accredited Test Lab Company
Page 120
If Samples drawn by ATL: If Samples drawn by Company:
_____________________ ______________________________
_________ (Signature/Name/Designation of the
(Signature/Name/Desi representative, Tissue Culture Production Facility)
gnation Stamp/Date of
Authorized person
of/by Accredited Test
Laboratory)
In the presence of: In the presence of:
_____________________
_____
(Signature/Name of ______________________________
authorized person from (Signature/Name of authorized person from Tissue
Tissue Culture Culture Production Facility)
Production Facility)
6. Particulars of payment of testing fees:
i. Amount in Rs:
ii. Demand
Draft/Banker’s
Cheque No./ Date of
Issue:
iii. Bank Name/Branch:
7. Any additional
information:
For Office (Testing Facility) Use

Date of receipt of
application:
Check list Status Scrutinized by
Application complete Yes No
Sample Received Yes No
with appropriate
quantity
Payment of Fees Yes No
Action Taken:
---------------------------------------------
(Signature/Name of
Director or HOD of
Accredited Test
laboratory/Date)
* Please indicate the type of tissue being sent for testing and its source. For example tissue could be leaf,
stem, rhizome, tuber from a field grown plant identified as mother plant or it could be from tissue culture.
In case cultures indicate stage of culture cycle.
** Sample registration number: Unique 20 digits code number consists of following digits
3 digits for registration number of tissue culture production facility /2 digits for plant species as listed at
Appendix 1/2 digits for variety/2 digits for registration number of ATL /6 digits for date/month/year of
sample received by ATL/1 digit for nature of sample/1 digit for mother plant/stock culture/last three digits for
Page 121
sample no. . Sample registration number will be entered by accredited test laboratory and will be
maintained throughout testing.
Code to be assigned to different commercially important plant species

Code No. Plant Species Code No. Plant Species
01 Aloe Vera 41 Philodendron
02 Alpine 42 Pineapple
03 Alstroemeria 43 Pointed Gourd
04 Anthurium 44 Pongamia
05 Apple 45 Populus
06 Bamboo 46 Potato
07 Banana 47 Rose
08 Black Pepper 48 Sandalwood
09 Calathea 49 Spathiphyllum
10 Cardamom 50 Stevia
11 Carnation 51 Strawberry
12 Casuarina 52 Sugarcane
13 Cattleya 53 Syngonium
14 Chrysanthemum 54 Teak
15 Citrus 55 Turmeric
16 Coccinea 56 Vanda
17 Cordylines 57 Vanilla
18 Cymbidium 58 Yucca
19 Dahlia 59 Zantedeschia
20 Date Palm 60 Others
21 Dendrobium
22 Eucalyptus
23 Ficus
24 Fig
25 Gerbera
26 Ginger
27 Gladiolus
28 Grape
29 Gypsophyllum
30 Hosta
31 Jatropha
32 Lavander
33 Lemon
34 Lilium
35 Limonium
36 Mangium
37 Neem
38 Paphiopedilum
39 Paulonia
40 Phalaenopsis
Note: List of varieties for each species (and its code) to be finalized from the available information and
in consultation with companies & concerned ATL/research institutions and to be circulated to all
Accredited Test Laboratories.

Annexure-2B
Page 122
Application for (Virus/ genetic fidelity) Testing for Batch
Certification of Tissue Culture Raised Plants
1. Name and Address of the recognized

tissue culture production facility:
Certificate No.
3. Name of authorized person & contact
details (Telephone/ Fax/Mobile/E-Mail):
4. Date of Sampling:
5. Number of samples drawn *:
6.1 Please indicate the 20 digits sample registration number of the plant tissue/stock
culture from which the batch has been derived in case the testing has been done under
NCS-TCP:
6.2. If testing of plant tissue/stock culture has not been done under NCS-TCP, give the
details of indexing lab
(please enclose a copy of test report)

7. Details of tissue culture plants required to be sampled: (Each sample should have at
least 0.5gm tissue/virus/test for all the known virus to be tested*)
**40 Batch Registration Number (to be filled by ATLs)
20 digits sample reg. 4 digits for batch number + 4 digits for

Plant species
no. from which batch batch size + 3 digits for number of

Stage of TC
Batch size
Batch No.
is derived samples
Variety
+ 6 digits for date, month and year of

sample received for batch certification
+ 1 digit for stage (ex-agar/hardened
)+2 digits for ATL
Plants
(20 digits)
Total 40 digits for batch registration number
(20 digits sample

reg. no. from
which batch is
derived)
8. Purpose of [ ] Domestic market

testing/certification: [ ] Export
Tick out in appropriate box
9. Testing For: Virus Indexing [ ] Genetic Fidelity [ ]
10. Sample drawn by: Accredited Test Laboratories [ ] Company [ ]
If Samples drawn by ATL: If Samples drawn by company:
________________________ __________________________
Page 123
(Signature/Name/Designation (Signature/Name/Designation of the representative,
Stamp/Date of Authorised Tissue Culture Production Facility)
person of/by Accredited Test
Laboratory)
In the presence of: In the presence of:
_______________________ ______________________________
(Signature/Name of (Signature/Name of authorized person from
authorized person from Tissue Tissue Culture Production Facility)
Culture Production Facility)
11. Particulars of payment of testing fees:

Amount in Rs:
Demand Draft/Banker’s
Cheque No./Date of Issue:
Bank Name/Branch:
For Office (Testing Facility) Use
Date of receipt of application:
Check list Status Scrutinized by
Application complete Yes No
Sample received in appropriate Yes No
quantities
Payment of Fees Yes No
Action Taken:
---------------------------------------------
(Signature/Name of
Director or HOD of
Accredited Test
laboratory/Date)
** Batch Registration Number will be entered by Accredited test laboratory and will
be maintained through out certification.
*Sampling method for TC raised plants
Batch size Number of tissue culture plants to be sampled
(sample size)
Up to 1000 Nos 1% plants subject to a minimum of 10 Nos
1001 to 10000 Nos 0.5% of plants subject to a minimum of 10 Nos
10001 to 100000 Nos 0.1% of plants subject to a minimum of 50 Nos
** Batch Registration Number:
The initial 20 digits of the batch registration number would be the sample registration number of the plant
tissue/stock culture from which the batch has been derived. Additional 20 digits would consist the following
format
4 digits for batch number + 4 digits for batch size + 3 digits for number of samples
+ 6 digits for date, month and year of sample received for batch certification + 1 digit for stage (ex-
agar/hardened +2 digits for ATL
Page 124
Annexure- 3
Recognized Tissue Culture Production Units
Till date 82 commercial Tissue Culture Production Units have been
Recognized” by the Department of Biotechnology (DBT), Govt. of India under the
“National Certification System for Tissue Culture Raised Plants (NCS-TCP)”..
These are
1. ACE Agro Technologies, Secunderabad
2. ACE Nurseries Pvt Ltd, Pune
3. Aditya Biotech Lab & Research Pvt Ltd, Raipur
4. AG Bioteck Laboratories (India) Ltd, Hyderabad
5. Agri Vitro Tech Laboratories, Secunderabad
6. Ajeet Seeds Ltd, Aurangabad
7. Anantha Biotechnologies, Ananthapur
8. Arcadia Agro, Mogar Anand
9. Aryave Biotech Pvt Ltd, Haridwar
10. ATGC Biotech, Raipur
11. Bhoomiputra Biotech, Jalgaon
12. Biotechnology cum commercial tissue Culture Centre, OUAT, Bhubaneswar, Odisha
13. Biotechnology Centre, Department of Horticulture, Govt of Karnataka
14. Boinchi Bioplants Pvt Ltd, Hooghly
15. BrookFileds Biotech Pvt Ltd, Kadapa
16. Cadila Pharmaceuticals Ltd(Agro Division), Ahmedabad
17. Central Plantation Crop Research' Institute, Kasargod, Kerala Coconut
18. Dept. of Biotechnology, Delhi University. Albizzia lebbeck, Acacia nilotica, Leucaena,
19. Devleela Biotechs, Raipur
20. Elegant Flower Company Pvt Ltd, Kolkata
21. Excel Plant Link Pvt Ltd, Bhubaneswar
22. Futura Bioplants Pvt Ltd, Pune
23. Gargi Bioteck Pvt Ltd, Pune
24. Genewin Biotech, Hosur
25. Godrej Agrovet Ltd(Plant Biotech Division), Secunderabad
26. Growmore Biotech Ltd, Hosur
27. Haryana Agril. University for Date palm
28. Hindustan Lever Lab for Coconut
29. HU Gugle Agro Biotech Co, Ahmadnagar
30. HU Gugle Biotech Pvt Ltd, Bangalore
31. In Vitro International Pvt Ltd, Bangalore
32. Indian Inst. of Science, Bangalore Sandal
33. Indo-American Hybrid Seeds Bangalore 3. BARC, Bombay Sandal
34. ITI Biotech Tissue Culture Lab, Betul
35. Jain Irrigation Systems Ltd Jalgaon
36. Kala Biotech Pvt Ltd, Pune
37. Kemrock Agritech Pvt Ltd, Vadodara
38. KF Bioplants Pvt Ltd, Pune
Page 125
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39. KF Biotech Pvt Ltd, Bangalore

40. Kore Biotech, Solapur
41. Krishiraj Nursery, Jalna
42. Kshitij Biotech Corporation, Karad
43. Kutch Crop Services Ltd, Kutch
44. La Chandra Tissue Culture Laboratory, Banaskantha
45. Lakshmi Biotech, Bangalore
46. Ligature Biotechnologies Pvt Ltd, Bangalore
47. LJ International Ltd (AVT), Kochi
48. Micropropagation Technology Park (TERI), New Delhi
49. Microsun Biotech, Secunderabad
50. MSR Biotech Pvt Ltd, Bangalore
51. National Bureau of Plant Genetic Resources, New Delhi Medicinal plants
52. National Chemical Laboratory, Pune Bamboo, Teak, Eucalyptus
53. Navsarjan Biotech, Bhuj, Kutch
54. Nirmeeet Biotech, Pune
55. PepsiCo India Holdings Pvt Ltd, Hoshiarpur
56. Ram Biotech, Jalgaon
57. Ramrich Biotech Pvt Ltd, Pune
58. Regional Plant Resource Centre, Nayapalli, Bhubaneswar
59. Reliance Life Sciences Pvt Ltd, Mumbai
60. Rise N Shine Biotech Pvt Ltd, Pune
61. Sagar Agrisciences Pvt Ltd, Barabanki
62. Sai Lara Biotechnologies, Hyderabad
63. Sarjan Biotech Pvt Ltd, Bhuj-Kutch
64. Secon Agriventures Pvt LTd, Bangalore
65. Seema Biotech, Kolhapur
66. Shaili Biotech (P) Ltd, Ahmedabad
67. Sheel Biotech Ltd, Gurgaon
68. Shree Biotech, Pune
69. Shri Ramco Biotech, Bangalore
70. Shriram Prathishtan Mandal’s Lokmangal Tissue Culture Lab, Solapur
71. Siddhi Plantek, Anand
72. SPIC Agro Biotech Centre, Coimbatore
73. Sri Soma Biotech, Guntur
74. Sristi Agro Biotech Pvt Ltd, Howrah
75. Sun Agrigenetics Pvt Ltd, Vadodara
76. Synergy Agri Products Pvt Ltd, Durgapur
77. Technico Agri Sciences Ltd, Chandigarh
78. Tissue Culture Facility, Biotech Park, Lucknow
79. Vasant Biotech, Yavatmal
80. Vasantdada Sugar Institute, Pune
81. Vasudha Biotech, Guntur
82. Vitroplant, Hyderabad
Page 126

Practocal Manual On Plant Tissue Culture

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Practocal Manual

Dr. Kailash Ch. Samal

Department of Agricultural Biotechnology

Dr. Kailash Ch. Samal

Department of Agricultural Biotechnology

Copyright 2018 Orissa University of Agriculture and

All rights reserved. No part of this publication may be reproduced,

Plant Tissue Culture

Traditionally, plants are multiplied by means of seeds (sexual propagation)

1.1. Various stages of micropropagation

Stage 0 : Selection and preparation of the mother plant

: Sterilization of the plant tissue takes place

Stage I : Initiation of culture

: Explant placed into growth media

: Explant transferred to shoot media; shoots can be constantly

Stage III : Rooting

: Explant transferred to root media

Stage IV : Transfer to green house and net house

: Gel washed TC plants were transferred to green house in

Stage V : Transfer to soil

: Explant returned to soil; hardened off

Practical Manual on Plant Tissue Culture and its Certification

1.4. Applications of plant tissue culture

1.5. Commercial prospects of Tissue culture in Agriculture

1.7. Highlights of the market survey on TCPs:

Tissue Culture Laboratory

2.1. Laboratory set up

 Washing and storage of glass wares

2.1.1. Washing room

2.1.2. Media room

2.1.3. Media storage room

2.1.4. Inoculation room

2.1.5. Culture room

2.2. Basic requirements

2.2.2. Inorganic macro nutrients

2.2.3. Inorganic Minor nutrients

2.2.3. Iron Sources

2.2.4. Organic supplement (Vitamins)

2.2.4. Carbon Sources

2.2.5. Plant growth regulators

2.3.1. Stock solution and media preparation

2.3.2. Nutrient stock solutions

2.3.3. MS macro (MS-I, 20X)

2.3.5. MS iron (MS-III, 20X)

2.3.6. MS Vitamins (MS- IV, 100X)

Myo-inositol 10.0g Dissolve all Use 10 ml

2iP 203.3 1N NaOH 0.1-0.5 Autoclave

6-Furfuryl amino- Deep Deep

Filter Deep Deep

2.3.11. 6-benzyl-aminopurine (BA) (0.1mg/ ml)

Finally volume make up

2.3.13. Preparation of media

2.3.14. Stock solution mixing

2.3.16. Pouring and Autoclaving

2.4. Initiation of Tissue Culture

2.4.1. Selection of Explants

2.4.2. Explants cleaning and treatment

Table 3: Types of surface sterilants used for aseptic culture

2.4.3. Inoculation of explants