REQUIREMENT OF PTC LAB and AN

INTRODUCTION TO MASS
MULTIPLICATION PROCEDURE FOR TISSUE
CULTURE

Dr. Deepak Sharma

JECRC, Jaipur
 INTRODUCTION

 Tissue culture is the in vitro aseptic culture of cells,

tissues, organs or whole plant under controlled
nutritional and environmental conditions often to
produce the clones of plants.
 The resultant clones are true-to type of the selected
genotype.
 The controlled conditions provide the culture an
environment conducive for their growth and
multiplication. These conditions include proper supply
of nutrients, pH medium, adequate temperature and
proper gaseous and liquid environment.
 MASS MULTIPLICATION
 Plant tissue culture technology is being widely used for large scale
plant multiplication.
 Apart from their use as a tool of research, plant tissue culture
techniques have in recent years, become of major industrial
importance in the area of plant propagation, disease elimination,
plant improvement and production of secondary metabolites.
 Small pieces of tissue (named explants) can be used to produce
hundreds and thousands of plants in a continuous process. A
single explants can be multiplied into several thousand plants in
relatively short time period and space under controlled conditions,
irrespective of the season and weather on a year round basis.
 Endangered, threatened and rare species have successfully been
grown and conserved by micropropagation because of high
coefficient of multiplication and small demands on number of
initial plants and space.
STAGES IN THE DEVELOPMENT OF TISSUE CULTURE
PROCESS FOR MASS MULTIPLICATION
Selection of superior clone

Collection of young branches and shoots

Introduction of Explants for in vitro culture

Shoot development from responsive explants

Elongation and Rooting stage

Multiplication of plantlet
MASS MULTIPLICATION PROCEDURE
 NEED
TO MULTIPLY IN VITRO?
Tissue culture has several critical requirements:
 Optimal protocol for mass multiplication
 Appropriate tissue (Explant)

 A suitable growth medium containing energy sources and

inorganic salts to supply cell growth needs. This can be liquid
or semisolid.
 Aseptic (sterile) conditions, as microorganisms grow much
more quickly than plant and animal tissue and can over run
a culture.
 Growth regulators - both auxins & cytokinins.

 Frequent subculturing to ensure adequate nutrition and to

avoid the build-up of waste metabolites.
 APPROPRIATE TISSUE (EXPLANT)
 Cell, tissue or organ of a plant that is used to start in
vitro cultures. Many different explants can be used for
tissue culture, but auxillary buds and meristems are
most commonly used.
 The explants must be sterilized to remove microbial
contaminants. This is usually done by chemical surface
sterilization of the explants with an agent such as
bleach at a concentration and for a duration that will kill
or remove pathogens without injuring the plant cells
beyond recovery.
 After tissue injury during dissection, such compounds will
be oxidized by polyphenol oxidases →tissue turn
brown/black.
 Phenolic products inhibit enzyme activities and may kill
the explants
Methods to overcome browning:
o Adding antioxidants [ascorbic acid, citric acid, PVP

(polyvinylpyrrolidone), dithiothreitol], activated charcoal

or presoaking explants in antioxidant
o Incubating the initial period of culturing in reduced

light/darkness
o Frequently transfer into fresh medium
 NUTRITION MEDIUM
 When an explants is isolated, it is no longer able to receive
nutrients or hormones from the plant, and these must be
provided to allow growth in vitro.
 In addition, the culture must be provided with the ability to
excrete the waste products of cell metabolism. This is
accomplished by culturing on or in a defined culture medium
which is periodically replenished.
 A nutrient medium is defined by its mineral salt composition,
carbon source, vitamins, plant growth regulators and other
organic supplements.
 pH determines many important aspects of the structure and
activity of biological macromolecules. Optimum pH of 5.0-6.0
tends to fall during autoclaving and growth.
MINERAL SALT
 MINERAL SALT COMPOSITION

 Macroelements: The elements required in concentration

> 0.5 mmol/l
 The essential macroelements: N, K, P, Ca, S, Mg, Cl

 Microelements: The elements required in conc. < 0.5

mmol/l
 The essential microelements: Fe, Mn, B, Cu, Zn, I, Mo, Co

 The optimum concentration →maximum growth rate

 CARBON SOURCES AND VITAMINS

 Sucrose or glucose (sometimes fructose), concentration

2-5% (20-40 g/l usually)
 An absolute requirement for vitamin B1 (thiamine)

 Growth is also improved by the addition of nicotinic

acid and vitamin B6 (pyridoxine)
 Some media contain pantothenic acid, biotin, folic acid,
p-amino benzoic acid, choline chloride, riboflavine and
ascorbic acid (C-vitamin)
 PLANT GROWTH REGULATORS
 Auxins: - induces cell division, cell elongation, swelling
of tissues, formation of callus, formation of
adventitious roots.
 - inhibits adventitious and auxillary shoot formation

 - 2,4-D, NAA, IAA, IBA,

 Cytokinins:- shoot induction, cell division

 - BAP, Zeatin, Kinetin, TDZ, 2iP

 Gibberellins: plant regeneration, elongation of

internodes -GA3…
 Abscisic acid: induction of embryogenesis -ABA
 OTHERS
 Media formulations
 Natural complexes (undefined)
 Gelling agent and supporting system
 Charcol
 Oraganic acid
 Antibiotics
 Amino acid / organic salts
 CELLULAR TOTIPOTENCY AND PLANT REGENERATION
 Unlike an animal cell, a plant cell, even one that highly
maturated and differentiated, retains the ability to
change a meristematic state and differentiate into a
whole plant if it has retained an intact membrane
system and a viable nucleus.
 Totipotency or Totipotent: The capacity of a cell (or a
group of cells) to give rise to an entire organism.
 Cultured tissue contain competent cells or cells capable
of regaining competence (dedifferentiation). e.g. an
excised piece of differentiated tissue or organ
 Dedifferentiation →callus

 Redifferentiation (whole plant) = cellular totipotency.

 1957 Skoog and Miller demonstrated that two
hormones affect explants’ differentiation:
– Auxin: Stimulates root development
– Cytokinin: Stimulates shoot development
 Generally, the ratio of these two hormones can
determine plant development:
–↑Auxin ↓Cytokinin = Root development
–↑Cytokinin ↓Auxin = Shoot development
– Auxin = Cytokinin = Callus development
 CLONAL PROPAGATION/MICROPRAPAGATION
 “… the art and science of multiplying plants in vitro.”
 Commercial production of plants through micropropagation
techniques has several advantages over the traditional
methods of propagation through seed, cutting, grafting and
air-layering etc. It is rapid propagation processes that can
lead to the production of plants virus free.
 The success of many in vitro selection and genetic
maniplation techniques in higher plants depends on the
success of in vitro plant regeneration.
 A large number of plants can be produced (cloned) starting
from a single individual
 Micropropagation allows the production of large
numbers of plants from small pieces of the stock
plant in relatively short periods of time. Depending on
the species in question, the original tissue piece may
be taken from shoot tip, leaf, lateral bud, stem or
root tissue.
 It is vegetative (asexual) methods of propagation which
can be used to produce 1,000,000 propagules in 6
months from a single plant.
 Stages in
Micropropagatio
n
 STAGES IN MICROPROPAGATION
 Micropropagation starts with the selection of plant
tissues (explants) from a healthy, vigorous mother plant.
Any part of the plant (leaf, apical meristem, bud and
root) can be used as explants.
Stage 0: Preparation of donor plant :
 Any plant tissue can be introduced in vitro. To enhance
the probability of success, the mother plant should be
ex vitro cultivated under optimal conditions to minimize
contamination in the in vitro culture
 Stage I: Initiation stage
 In this stage an explant is surface sterilized and transferred
into nutrient medium.
 Generally, the combined application of bactericide and
fungicide products is suggested. The selection of products
depends on the type of explants to be introduced.
 The surface sterilization of explant in chemical solutions is
an important step to remove contaminants with minimal
damage to plant cells. The most commonly used
disinfectants are sodium hypochlorite, calcium hypochlorite,
ethanol and mercuric chloride (HgCl2).
 The cultures are incubated in growth chamber either under
light or dark conditions according to the method of
propagation.
 Stage II: Multiplication stage
 The aim of this phase is to increase the number of
propagules [22]. The number of propagules is multiplied by
repeated subcultures until the desired (or planned) number
of plants is attained.
Stage III: Rooting stage
 The rooting stage may occur simultaneously in the same
culture media used for multiplication of the explants.
However, in some cases it is necessary to change media,
including nutritional modification and growth regulator
composition to induce rooting and the development of
strong root growth.
 Stage IV: Acclimatization Stage
 At this stage, the in vitro plants are weaned and
hardened. Hardening is done gradually from high to
low humidity and from low light intensity to high
light intensity.
 The plants are then transferred to an appropriate
substrate (sand, peat, compost etc.) and gradually
hardened under greenhouse.
 APPLICATIONS OF MICROPROPAGATION
 Screening cells rather than plants for advantageous
characters, e.g. herbicide resistance/tolerance.
 Large-scale growth of plant cells in liquid culture

 To cross distantly related species by protoplast fusion

and regeneration of the novel hybrid.
 Embryo rescue

 For production of doubled monoploid plants from

haploid cultures to achieve homozygous lines
 As a tissue for transformation, followed by either short-term
testing of genetic constructs or regeneration of
transgenic plants.
 In vitro conservation of germplasm
 ADVANTAGES OF MICROPROPAGATION
 Micropropagation offers several distinct advantages not possible
with conventional propagation techniques.
i. Rapid multiplication of genetically uniform plants
ii. The production of multiples of plants in the absence of seeds
or necessary pollinators to produce seeds
iii. The regeneration of whole plants from plant cells that have
been genetically modified.
iv. The production of plants in sterile containers that allows them
to be moved with greatly reduced chances of transmitting diseases,
pests and pathogens.
v. The production of plants from seeds that otherwisehave very
low chances of germinating and growing, e.g. orchids and
nepenthes.
vi. The production of plants throughout the year i.e. not affected by
environmental conditions.
 Flow chart
summarizin
g tissue
culture
experiment
s
 REFERENCE
 Akin-Idowu, P. E., Ibitoye D.O. and Ademoyegun, O.T. (2009). Tissue
culture as a plant production technique for horticultural crops. Afr. J.
Biotechnol. 8(16): 3782-3788.
 George, E.F. (1993). Plant propagation by Tissue Culture. Eastern
Press, Eversley.
 Hussain, A., Ahmed, I., Nazir, H., and Ullah, i., (2012). Plant Tissue
Culture: Current Status and Opportunities.
http://dx.doi.org/10.5772/50568
 Modi, A. R., Patil, G., Kumar, N., Singh, A. S., Subhash, N., (2012). A
Simple and Efficient In Vitro Mass Multiplication Procedure for Stevia
rebaudiana Bertoni and Analysis of Genetic Fidelity of In Vitro Raised
Plants Through RAPD. Sugar Tech (Oct-Dec 2012) 14(4):391–397
 Chaturvedi, H.C., Jain, M. and Kidwai, N. R. (2007). Cloning of
medicinal plants through tissue culture - A review. Indian journal of
experimental biology, 45: 937-948.
Thank You for ur attention