Presentaion on techniques of in vitro cultures

2. K. K. Wagh College of Agriculture, Nashik.
(Saraswatinagar, Panchavati, Nashik-3)
(Affiliated to M.P.K.V., Rahuri)
Dept. of Agricultural Botany

3. Techniques of in vitro cultures
Deshmukh Sharayu Khasherao
AKN-2013/26
Module In-charge
Prof. S. S. Kharade
Dept. of Agriculture Botany,
K. K. Wagh College of Agriculture,
Nashik

4. Experiential Learning Programme
Academic Year :- 2016-17
Module No :- AEL- BOT-489
Module Title :- Commercial Plant
Tissue Culture
Credit :- 20(0+20)

5. CONTENTS
1. Introduction
2. Important terms
3. Important steps in in vitro culture
4. Tissue culture techniques
• Organ culture
• Seed culture
• Meristem culture
• Embryo culture

6. • Ovary culture
• Anther culture
• Callus culture
• Cell suspension culture
• Protoplast culture
• Applications

7. INTRODUCTION
Plant cells divide indefinitely in appropriate culture
medium and regenerate into whole plant .
Tissue culture refers to the growing of living plant cells,
tissues or organs on suitable nutrient medium in sterile
conditions.
For in vitro culture, explants from healthy and young part
of the plant is used to regenerate the whole plant.

8. Important features of in vitro
techniques:-
1. The in vitro works are carried out in a test
tube, dish or flask i.e. outside the living
organism. Hence in vitro techniques are also
referred to as plant tissue culture techniques.
2. Tissue culture work is confined to plants.
3. The tissues are grown in suitable nutrient
medium.
4. Tissues are grown outside the parent
organism from which tissues have been taken.

9. IMPORTANT TERMS
Various terms are used to define the phenomenon in in
vitro studies. The following terms are :
Differentiation: It is defined as the process by which
meristematic cells are converted into two or more types of cells,
tissues or organs which are different from each other.
De-differentiation: The term is used to denote the process of
formation of unorganised tissues from the highly organised
tissues.
Re-differentiation: The dedifferentiated cells lose the capacity
of division and becomes mature to form specific functions. Such a
tissue is said to be redifferentiated and process is known as re-
differentiation.
Regeneration: Formation of an entire plant form cultured
explants directly or via callus indirectly is called regeneration.

10. Organogenesis: Formation of adventitious roots or shoots
from cells is called as organogenesis
Totipotency: The phenomenon of regeneration of plant cell to
develop into whole plant.
Embryogenesis:Embryogenesis starts from a
single embryogenic cell, that can be a zygote (the product of the
fusion of an egg and a sperm during fertilization), or an
undifferentiated callus cell. Embryos developing from zygotes are
called zygotic embryos, while those derived from somatic cells
are called somatic embryos.

11. Differentiation
De-differentiation
Re-differentiation

12. Regeneration Embryogenesis
Organogenesis

13. Important steps in in vitro culture
In vitro culture technique generally consists of four main steps, they
are:
Isolation of tissues
Regeneration and callus formation
Embryogenesis
Organogenesis
Isolation of tissues: Tissues for regeneration can be isolated with the help of
sterilized blade from any plant part i.e. leaf, stem, bud etc. The isolated tissues are
sterilized and then grew on culture medium. Tissues should be isolated from diseased
free portion.
Regeneration and callus formation: Tissues proliferate on the callus medium
and give rise to a mass of cells called callus.
Embryogenesis: The process of formation of somatic embryos from the callus is
called embryogenesis. Sometimes somatic embryos are not formed somatic buds are
formed which germination gives rise to plant.
Organogenesis: The process of differentiation of shoot or root from the somatic
embryos is called organogenesis. Sometimes, a complete plant develops directly from
the somatic bud. The plants thus obtained are transferred after sometime to pot culture
from culture medium.

14. TISSUES
Culture medium
Proliferation
Callus formation
Germination
New plant
Transfer to pot culture
Somatic
embryo
Callus
buds
Ref. Introduction to biotechnology.
By- Pundhan Singh

15. TISSUE CULTURE TECHNIQUES
In vitro techniques give rise to new plants by regeneration of
tissues and organs in the nutrient medium. Various plant
parts like meristem, embryo, anther, cells, ovule etc are used
for regeneration. Depending upon the plant parts used tissue
culture techniques are of following types:
Plant
Tissue
Culture
Embryo
culture
Callus
culture
Organ
culture Bud
culture
Cell culture
Protoplast
culture
Meristem
culture
Seed
culture

17. 1.Organ culture
Culturing isolated organs or tissues such as roots, stem,
or leaf in an artificial media under controlled conditions is
known as organ culture.
Any plant organ can serve as an explant to initiate
cultures
No. ORGAN CULTURE
1. Shoot Shoot tip culture
2. Root Root culture
3. Leaf Leaf culture
4. Flower Anther/ ovary/ ovule
culture

18. Importance of Organ Culture
•It provides an excellent experimental system to define the
nutrients and growth factors normally received by the organ from
other parts of the plant body and from its external environment.
•It is valuable in studies of the interdependence of organs for
growth hormones and other growth factors.
•It opens up a new avenue for the developments in agriculture and
horticulture.

19. Types of Organ Culture.
On the basis of explant, organ culture can be classified into
different types. Each type of organ has its own particular
advantage and importance for specific research project.
ORGAN
CULTURE
Vegetative
organs
Reproductive
organs
Root
culture
Leaf
culture
Shoot-
tip
culture
Complete
flower
culture
Isolated
pollen
culture
Isolated
ovule and
embryo
culture

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

21. 3.Meristem culture
Meristem culture is the in vitro culture of a generally shiny
special dome like structure measuring less than 0.1mm in
length .
Ref. Plant tissue culture
By Kalyan Kumar De

22. The excised meristem can be cultured aseptically on agar
medium and under appropriate condition will grow out
directly into a small leafy shoot or multiple shoots.
Alternatively, the meristem may form a small callus on
which large number of shoots will develop.
Cytokinins in the nutrient medium plays an important role
for the development of shoots from meristem.
BAP is the most effective cytokinin commonly used in
meristem culture.

23. Importance of Meristem culture.
The uses of meristem culture mainly include:
Virus eradication
Micro-propagation and
Storage of genetic resources.
1. Virus eradication.
• Many plants contain viruses which reduce their
potential yield and quality. It is therefore important to
produce virus free stocks which can be multiplied.
• Generally meristematic tissues are free from viruses
due to their high mitotic activity hence vascular tissues
are not developed in the meristem region. Therefore
Meristems are ideal explants to produce virus free
stock.

24. 3. Storage of genetic resources.
• Few seeds are not accepted for storing as a genetic
resource.
• So , the meristem from such plants can be stored in
vitro.
2. Micro-propagation.
• A sexual or vegetative propagation of whole plants using
tissue culture technique is known as Micropropagation.
• Meristems of plants can successfully be used for
micropropagation.

25. 4. Embryo culture
The embryo of different developmental stages,
formed within a female gametophyte through
sexual process, can be isolated aseptically from
the bulk of maternal tissues of ovule, seed or
capsule and cultured in vitro under aseptic and
controlled physical conditions in glass containing
solid or liquid medium to grow directly into plantlet.

26. Aseptic excision of the embryo is carried out and it
is transferred to a suitable nutrient medium for
development under optimum culture conditions.
In case of smaller embryos it is important to remove
the embryo carefully without causing any injury.
The most important aspect of embryo culture work
is the crucial selection of medium necessary to
sustain continued growth of the embryo.
In culture embryos are not induced to form callus
tissue but they are allowed to form a plantlet.

27. Ref. Plant tissue culture
By Kalyan Kumar De

28. Importance of embryo culture
•It helps in analysis of various parameters of embryonic
growth.
•This culture helps to understand the control of
differentiation and the nutritional requirements of
progressively smaller embryos.
•The culture of embryos has facilitated to understand the
relationship of the different parts of embryo and its final
form in culture.
•Changes in nutritional requirement of embryos at different
stages can be demonstrated by embryo culture.

29. 5. Ovary culture
Ovary culture is a technique of culture of ovaries
isolated either from pollinated or unpollinated
flowers
Ovaries grow in culture and form the fruits that ripen
and produce viable seeds.
This development takes place on a simple nutrient
medium containing only mineral salts and sucrose.
But the ovaries of unpollinated flowers do not grow
on simple nutrient medium. However, use of some
synthetic auxins such as 2,4-D, 2,4,5-T in the nutrient
medium induces the development of ovaries of
unpollinated flowers.

30. Ref. Plant tissue culture
By Kalyan Kumar De
Importance of ovary culture
•It is important to study the early
development of embryo, fruit,
different aspects of fruit physiology
including respiration, maturation and
disease.
•Role of floral organs can be studied
from the in vitro culture of ovary.
•Ovary culture in vitro fertilization has
been used to eliminate the obstacles
such as failure of pollen germination
on stigma or the slow and insufficient
growth of pollen tube as well as
abscission of flowers.

31. 6.Anther culture
Anther culture is a technique by which the developing anthers at a
precise and critical stage are excised aseptically from unopened
flower bud and are cultured on a nutrient medium where the
microspores within the cultured anther develop into callus tissue or
embryoids that give rise to haploid plantlets either through
organogenesis or embryogenesis.
Anther culture helps in the production of haploid plantlets.
The principle behind the anther culture is that without disturbing the
natural habitat and environment of enclosed anther, pollen can be grown
by culturing the anther.
In culture conditions the tissues of anther will remain living and will
encourage the development of pollen by nursing and providing nutrient.
Induction of haploids can be enhanced by keeping the anther or flower
bud at low temperature.

33. Importance of anther culture
•It is an efficient way for the production of
haploids from the microspores present
within the anther.
•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.

34. 7.Callus culture.
Callus is the unorganised mass of cells produced from isolated
plant cells, tissues or organs when grown aseptically an artificial
nutrient medium under controlled conditions.
The plant parts are first washed with liquid detergent,
then surface sterilized by most common chemicals such as
mercuric chloride or sodium hypochlorite for limited time.
The surface sterilized plant material is cut into small
segments.
Then explants are transferred on suitable nutrient
medium solidified with agar.
Once the growth of the callus tissue is well established,
portions of the callus tissue can be removed and
transferred onto fresh nutrient medium to continue growth.

35. Ref. Plant tissue culture
By Kalyan Kumar De

36. Importance of callus culture.
•The whole plant can be regenerated in large number
from callus tissue through manipulation of the nutrient
and hormonal constituents in the culture medium.
•Callus tissue is a good source of genetic variability,
so it is possible to regenerate a plant from genetically
variable cells of callus tissue.
•Cell suspension culture in moving liquid medium can
be initiated from callus culture.
•To obtain commercially important secondary
metabolites.

37. 8.Cell suspension culture
Cell suspension culture is a type of culture in which single
cells or small aggregates off cells multiply while suspended
in liquid medium. It is also referred to as cell culture or
suspension culture.
The cultivation of free cells as well as small cell aggregates in
a chemically defined medium as a suspension was initiated to
study the morphological and biochemical changes during their
growth and developmental phases.
To achieve an ideal suspension culture, most commonly a
friable callus is transferred to liquid medium where it breaks up
and readily disperses.
After eliminating the large callus pieces, only single cells and
small cell aggregates are again transferred to fresh medium and
after two or three weeks a suspension of actively growing cells is
produced.

38. Ref. Plant tissue culture :By Kalyan Kumar De
Growth patterns in suspension culture.
The growth curve for a
typical suspension culture
consists of:
Lag phase
Logarithmic phase
Linear phase
Stationary phase

40. Importance of cell suspension culture
Cell suspension technique is capable of contributing
many significant informations about cell physiology,
biochemistry, metabolic events at the level of individual cells
and small cell aggregates.
It is also important to understand the organ formation or
embroid formation starting from single cell.
Mutagenesis studies may be facilitated by the use of cell
suspension cultures to produce mutant cell clones from
which mutant plants can be raised.

41. 9.Protoplast culture
The protoplast is a part of plant cell which lies within the cell
wall and which can be isolated by removing the cell wall by
mechanical or enzymatic procedure, and culture of this
protoplast is known as protoplast culture.
The basic principle of protoplast culture is the aseptic
isolation of large number of intact living protoplasts removing
their cell wall and culture them on a suitable medium for
growth and development.
The isolated protoplast can be cultured on medium.
The cells from protoplast enter cell division which is
followed by the formation of callus.
Such callus consists the property of morphogenesis and
plant regeneration.

42. Ref. Plant tissue culture: by Kalyan Kumar De

43. Importance of protoplast culture
•Protoplast culture technique is important
because of far-reaching application in studies
of fundamental and applied research in
experimental biology, somatic hybridisation,
genetic engineering and somatic cell genetics.

44. Applications
Sr.
No.
Culture
Technique
Applications
1 Meristem Culture Virus free plant Production
2 Seed Culture Increase efficiency of Seed Germination
3 Callus Culture Produce Somatic Embryo
4 Embryo Culture
Embryo Rescue, Immature Seed, Seed
Sterility
5 Anther Culture Haploid Plant Production
6 Suspension Culture Single Cell Production
7 Protoplast Culture Somatic Hybrid Production

45. References
Plant tissue culture
By: Kalyan Kumar De
 Introduction to biotechnology
By: Pundhan Singh
Biotechnology and plant tissue culture
By: S.Chand