 Name :Chaitanya Nihalkumar Vankalas

 Course No. : VSC -503

 Course Title : Growth And Development Of Vegetable Crops

 Course Teacher : Prof. M.G. Agale
 College Name – Dr Sharadchandra Pawar Post Graduate Institute Baramati
 NAME OF TOPIC

TISSUE CULTURE TECHNIQUES IN VEGETABLE CROPS

 INTRODUCTION
Plant tissue culture broadly refers to the cultivation in vitro of all plant parts,
whether a single cell, a tissue or organ under aseptic conditions.
This new technique has enabled us to increase the knowledge in the following
field of studies.
 Totipotency, nutrition, metabolism, division, differentiation and preservation
of plant cells.
 Morphogenesis and plant regeneration from individual cells or tissues
through the process namely organogenesis or somatic embryogenesis. 
Variations generated through in vitro culture.
 Evolution of haploids through anther and pollen culture including ovule
culture.
 Wide hybridization programmes through ovule, ovary and embryo
 cultures to overcome both pre zygotic and
post zygotic sterility mechanisms
 Micropropagation of plant materials
 In vitro selection of mutants tolerant to biotic
and abiotic stresses.
 In vitro culture and secondary metabolite
biosynthesis.
 Plant genetic engineering through in vitro
culture methods and DNA transfer technique.
Haberlandt G. – Father of plant
tissue culture.
 Techniques of in vitro cultures
 Various terms are used to define the phenomenon in in vitro studies. For example
differentiation, de-differentiation, re-differentiation, regeneration and morphogenesis are
terms with overlapping meanings. To give a clear cut view for the usage of terms, the
sharp differences among them exposed hereunder.
 Differentiation: The term differentiation is used in many different senses in biology. In
broad sense, it is defined as the process by which meristematic cells are converted into
two or more types of cells, tissues or organs which are qualitatively different from each
other.
 De-differentiation: The term is used to denote the process of formation of unorganized
tissues from the highly organised tissues.
 Re-differentiation: The process of differentiation occurring in an undifferentiated tissue.
 Regeneration: It is defined as the structuring of any part, which has been removed or
physiologically isolated from the organism. In other words, genesis of an entire plant
from cultured explants directly or via callus indirectly is called regeneration.
Morphogenesis
Organogenesis
 1)Micro-propagation-
 Explants used in micropropagation – Root tip ,shoot tip, axillary bud,
leaf tip , nodal base, apical meristem , lateral bud.
 Stages in micro -propagation
Stage 0: Preparative stage –
 To reduce the contamination problem in the subsequent stages, mother plant
should be grown in a glasshouse and watered so as to avoid overhead irrigati
Stage 1. Initiation of culture – A)Explant :
Explant used Objective
Pre formed vegetative bud For enhanced axillary branching,
Sub-millimeter shoot tips For virus-free plants

Nodal cuttings If Stock is virus tested

B) Sterilization
C) Browning Of Media
 Stage 2. Multiplication –
-Through callusing:
- Adventitious bud formation:
-Enhanced axillary branching:
 Stage 3. Rooting of shoots –
 Stage 4. Transplantation-
Diagrammatic representation of micro propagation stages
ADVANTAGES of micropropagation Disadvantages of micropropagation
1. Clonal mass propagation - extremely large 1.
numbers of plants can be produced. Rather than 2.
getting 10000 plants per year from an initial cutting in 3.
vegetative propagation, one can obtain more than 4.
1,000,000 plants per year from one initial explant
through micropropagation. 5.
2. Culture is initialized from small parts of plants – so
no need of much space: from 1 m2 space in culture 6.
room, 20000 - 100000 plants can be produced per
year. 7.
3. Production of disease and virus free plantlets. This
leads to simplification of international exchange of 8.
plants 9.
4. Micropropagation enables growers to increase the
production of plants that normally propagate very
slowly such as narcissus and other bulbous crops.
6. Vegetative propagation of sterile hybrids can be
used as parent plants for seed production. Eg.
Cabbage.
7. In vitro cultures can be stored for long time through
cryopreservation. 9. Breeding cycle can be shortened.
Expensive laboratory equipment and service
No possibility of using mechanization
Plants are not autotrophic
Poor acclimatization to the field is a common
problem (hyperhydricity)
Risk of genetic changes if 'de novo' regeneration
is used.
Mass propagation cannot be done with all crops
to date. In cereals much less success is achieved
regeneration is often not possible, especially with
adult woody plant material.
More problems in inducing rooting.
May not get uniform growth of original plant
from tissue culture. Each explant has different in
vitro growth rates and maturation. Thus cannot be
used for floriculture crop production where
uniformity is critical.
 Horticultural uses for plant tissue culture
1. Clonal mass propagation. The important point here is that extremely large
numbers of plants can be produced. Rather than getting 10000 plants per year
from an initial cutting, one can obtain upwards of 1,000,000 plants per year from
one initial explant.
2. Difficult or slow to propagate plants. Micropropagation enables growers to
increase the production of plants that normally propagate very slowly such as
narcissus and other bulbous crops.
3. Introduction of new cultivars eg. Dutch iris. Get 5 daughter bulbs annually.
Takes 10 years for commercial quantities of new cultivars to be built up. Can get
100-1000 bulbs per stem section.
4. Vegetative propagation of sterile hybrids used as parent plants for seed
production. Eg. cabbage.
5. Pathology - Eliminate viruses, bacteria, fungi etc. Use heat treatment and
meristem culture. Used routinely for potatoes, carnation, mum, geranium, garlic,
gypsophila.
Types of cultures
Organ cultures: Culturing isolated organs or tissues such as roots, stem, or leaf in an artificial media under controlled
conditions are known as organ culture. Depending on the type of organs or tissue used for establishing the culture, organ cultures are
named accordingly.
The following are the various types of organ culture and its specific purpose:

Seed culture: Increasing the efficiency of germination of seeds that are difficult to germinate in vivo, precocious germination by
application of plant-growth regulators, and production of clean seedlings for explants or meristem culture.

Embryo culture: Overcoming embryo abortion due to incompatibility barriers, overcoming seed dormancy and self-sterility of
seeds, and embryo rescue in distant (interspecific or intergeneric) hybridization where endosperm development is poor, shortening of
breeding cycle, etc.

Ovary or ovule culture: A common explant for the initiation of somatic embryogenic cultures, for the production of haploid
plants, overcoming abortion of embryos of wide hybrids at very early stages of development due to incompatibility barriers, and in
vitro fertilization for the production of distant hybrids avoiding style and stigmatic incompatibility that inhibits pollen germination
and pollen tube growth.

Anther and microspore culture: Production of haploid plants, production of homozygous diploid lines through
chromosome doubling, thus reducing the time required to produce inbred lines, and for uncovering mutations or recessive phenotypes.

Explant culture Explant culture is actually the tissue culture. Culturing of any excised tissue or plant parts such as leaf tissue,
stem parts, cotyledon, hypocotyls, root parts, etc., is called explant culture.
 EXAMPLES
Production of haploid (n) plant through anther/microspore culture in
horticultural crops
 Crop species Mode of haploid development
 Asparagus officinalis Direct/indirect androgenesis
 Beta vulgaris Direct/indirect androgenesis
 Brassica oleracea Direct/indirect androgenesis
 Capsicum annuum (Fresh World Farms® Sweet Mini- Anther culture-derived variety released for
pepper developed by DNAP Holding Corporation) commercial cultivation
 Cucumis sativus Indirect androgenesis
 Lycopersicon esculentum Direct/indirect androgenesis
 Raphanus sativus Direct androgenesis
 Solanum tuberosum Direct/indirect androgenesis
 S. melongena Direct/indirect androgenesis
 REFERENCES
Bhojwani, S.S. and Rajdan, M.K. 2004. Studies in Plant Science-5, Plant Tissue Culture: Theory and
Practice: A Revised Edition. Elsvier Science BV, The Netherlands.

Cassells, A.C. and Bajaj, Y.P.S. 1991. Setting up a commercial micropropagation laboratory.
Biotechnology in Agriculture and Forestry vol. 17. Springer-Verlag; Berlin, Germany.

De Fossard, R.A. and de Fossard, H. 1988. Coping with microbial contaminants and other matters in
a small commercial micropropagation laboratory. Acta Hort. 225 : 167-176.

Dixon, A. and Gonzalez, R.A. 1994. Plant Cell Culture: A practical manual. IRS Press, Oxford, UK.

George, E.F 1993. Plant Propagation by Tissue Culture, Part I & II. Exgetics Limited, Edington, UK.

Murashige, T. 1974. Plant propagation through tissue culture. Ann. Rev. Plant Physiol. 25 : 135-166.

Singh, B.D. 2004. Biotechnology: Expanding Horizons. Kalyani Publishers, New Delhi.

Singh, B.D 2005. Plant Biotechnology. Kalyani Publishers, New Delhi

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