1. Tissue Culture Technology
By A.Arputha Selvaraj

2. Molecular Biology Applications in Plant Breeding
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1. PLANT CELL AND TISSUE CULTURE
a. Procedures Utilizing Tissue Culture Techniques
b. Tissue Culture Techniques
c. Plantlet Regeneration
2. CLONAL PROPAGATION VIA TISSUE CULTURE
Commercial Applications
i. PROPAGATION OF DISEASE-FREE GENETIC STOCKS
ii. FREEZE PRESERVATION OF GERM PLASM
3. EMBRYO CULTURE, OVULE CULTURE, IN VITRO
POLLINATION
a. Embryo Culture
b. Ovule Culture
c. In Vitro Pollination and Fertilization

3. Molecular Biology Applications in Plant Breeding
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4. ANTHER CULTURE AND HAPLOID PLANT PRODUCTION
a. Anther Culture Procedures
b. Factors Affecting Haploid Plant Production through Anther
Culture
c. Utilization of Anther Culture Derived Doubled-Haploids in Plant
Breeding
5. GENETIC VARIABILITY FROM CELL CULTURES:
SOMACLONAL VARIATION
6. SOMATIC CELL HYBRIDIZATION
7. PLANT GENETIC ENGINEERING
8. MOLECULAR MARKERS

4. Introduction to Plant Tissue Culture
• Plant cell and tissue culture includes a wide range of
cultural techniques for regeneration of functional
plants from embryonic tissues, tissue fragments, calli,
isolated cells, or protoplasts
• Basic Concept is Totipotency :
each living sell of a multicellular organism would be
capable of developing independently if provided with
the proper external condition (white, 1954)
totipotent cell is one that is capable of developing by
regeneration into a whole organism (morgan, 1901)

5. Micropropagation Laboratory
The Stages Are:
• Medium Preparation and Explant Selection
• Establishment of aseptic culture
• Proliferation
• Rooting
• Acclimatization

6. Basic Tissue Culture Procedures

7. Procedures Utilizing Tissue Culture Techniques
Clonal propagation: the rapid multiplication of genetic stocks, through
tissue culture, including procedures for isolation of pathogen-free plant
materials and freeze-preservation of germplasm;
Embryo and ovule culture: the rescue and propagation on a sterile
nutrient medium of immature embryos from interspecific or intergeneric
crosses;
Anther culture: the culturing of anthers in vitro for the purpose of
generating haploid plantlets;
Somaclonal variation: genetic variation induced in somatic cells cultured
in vitro;
Somatic cell hybridization: the fusion of protoplasts from genetically
diverse germplasms; and
Genetic engineering (transformation): In plants, the transfer of DNA
from a donor species to a recipient species by means of a bacterial
plasmid, virus, or other vector, or through microinjection or biolistic
device. Plants receiving the new DNA are said to be transformed, and
are regarded as transgenic plants.

8. Tissue Culture Techniques; Definitions and examples
It is the culture of isolated plant cells or detached fragments of
plant tissue on a nutrient medium under aseptic conditions and
their subsequent regeneration into functional plants
Undifferentiated plant cells often can be made to develop into
functional plants when appropriately cultured in vitro. This
property is designated totipotency. The need to regenerate plants
from totipotent cells makes plant cell and tissue culture an
essential step in utilization of the new molecular technology.
Model species, egs.; tobacco, potato, alfalfa, sugarcane, rice, and
various horticultural species, many of which are readily
propagated vegetatively.
Field crop species, eg.; corn, sorghum, forage grasses, cotton,
soybean, and the grain legumes, are difficult to regenerate.

9. Tissue Culture Manipulations

10. Factors Affecting Tissue Culture Efficiency
Plant regeneration from tissue culture varies with the
following parameters:
plant species,
genotype within the species,
source of the cultured tissue,
age and health of the donor plant,
nutrient medium,
other factors

11. In vitro Regeneration of Wheat
A: Shoot emergence from callus
B: Profuse shoot production
C: Regenerated wheat plantlet

12. What are explants and how are they obtained ?
Plant tissue cultures are generally initiated from
multicellular tissue fragments, called
explants, obtained from living plants.
Explants may originate from a wide range of plant
tissues, such as…
leaf,
stem,
root,
petiole,
hypocotyl,
cotyledon,
embryo, or
meristem

13. From “explants” to “callus” on a solid medium
The explant is commonly cultured on a nutrient medium solidified
in agar. Explants from most species of plants may be induced to
divide in an unorganized manner on specifically formulated
nutrient media
An undifferentiated mass of cells, known as callus (plural, calli), is
formed within 4 to 8 weeks.
The callus may be divided, with clusters of cells transferred to
fresh agar media to form subcultures. Repeated subculturing of
the callus permits rapid multiplication of the cultured material.
Plant regenerability may decline, and genetic stability of the plant
material may be altered, with successive subculturing.
Callus cultures are incubated under aseptic conditions, normally
in dim light, with temperatures around 25 C.

14. Nutrient medium and the role of growth hormones?
The nutrient medium commonly contains
inorganic salts, sugar as a source of carbon, and vitamins to maintain high
growth rates
Phytohormones such as auxins and cytokinins may be added to control cell
growth and division
The ratio of auxin to cytokinin has an important role in the initiation of
shoot and root primordia.
a low auxin: cytokinin ratio stimulates initiation of shoot buds and suppresses
root initiation;
a high auxin: cytokinin ratio leads to dedifferentiation and favors root
initiation;
an intermediate ratio favors continued division of cells as undifferentiated
callus
The optimum culture medium may vary with the species, the genotype
within the species, and the origin and age of the cultured tissue.
The preferred physical state of the culture medium, whether a liquid
medium or a solid agar gel, may vary with the species and the culture
environment.

15. Phytohormone
Structures

16. Plantlet Regeneration
Plantlets can be initiated
Indirectly from callus via
• adventitious shoots
• somatic embryos OR
Directly from explants such as
• Axillary buds
the culture is transferred to a rooting medium to induce root
initiation and subsequently plantlets
Somatic embryos have both root and shoot apices present and
can develop directly into plantlets.
Adventitious shoot initiation (organogenesis) occurs with a wider
range of plant species than initiation of somatic embryos; few
major field crop species can be routinely induced to form somatic
embryos.

17. “Establishment” of regenerated plantlets
Establishment: It refers to the successful growth and development of
plantlets regenerated through tissue culture techniques in soil.
The establishment of a healthy plantlet in soil with minimum mortality
is as essential for success in tissue culture propagation as obtaining a
high frequency of plantlet regeneration.
Difficulty in establishment: Species differ in their capability of
adjusting to the new environment. During this period the plantlet
must change from the heterotrophic state to the autotrophic state,
where it synthesizes its own organic food requirements.
Water loss from the regenerated plantlet is high, due to inadequacy of
the root system formed in culture to maintain the plant in soil, and a
reduced presence of epicuticular wax on leaves and stems of regenerated
plantlets. The regenerated plantlet must be protected from desiccation
and hardened to attain some tolerance to moisture stress.
the new plantlets, which have been developed under aseptic conditions,
should be protected from soil pathogens so that they can grow and
develop into healthy plants.

18. CLONAL PROPAGATION VIA TISSUE CULTURE
clonal propagation, cloning, or micropropagation: It is
the use of tissue culture technology for rapid
regeneration of particular plant genotypes.
Some potential uses of clonal propagation in
agronomic crops are:
large-scale increase of a heterozygous genotype,
increase of a self-incompatible genotype,
increase of a male-sterile parent in a hybrid-breeding
program,
propagation of disease-free genetic stocks, and
preservation and international exchange of germplasm.

19. Advantages of clonal propagation
Shoot tips cloned from axillary buds or meristem
tissue produce fewer genetic variants than cultures
from more mature tissues.
If, in addition to the meristematic region, one or two
leaf -primordia are included in the shoot-tip explant,
the explants will be larger, require less time for
excision, and have a higher survival rate than the
smaller explants cloned without the leaf -primordia.
Axillary shoots produced on the shoot-tip explants can
be subcultured until the required number of potential
plantlets are obtained. The plantlets are transferred to
a rooting medium and later transplanted into soil.

20. Commercial Applications of Clonal Propagation
Clonal propagation has the potential for propagation of thousands
of plantlets from a single genetic stock.
Examples:
orchids,
pyrethrum,
potato,
asparagus,
strawberry, and
various flowers or herbaceous ornamentals that set seed poorly.
This may not be suitable for seeding field crops
In vitro propagation may have application for early generation
increase of breeding materials in crop species with sparse seed-
setting, provided that efficient tissue culture procedures that can
be routinely employed have been developed for those species
and that genetic identity can be maintained in the plants
propagated.

21. Thanks