Introduction Advantages of Micropropagation over the conventional methods History Stages of Micropropagation 1. Stage 0; Preparative stage 2. Stage 1; Initiation of aseptic cultures A) Explant B) Sterilization C) Browning of medium Factors affecting initiation stage Conclusions References

1. Plant Tissue Culture
Culture Stage – 1; Initiation of
culture
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. SYNOPSIS
•Introduction
•Advantages of Micropropagation over the
conventional methods
•History
•Stages of Micropropagation
1. Stage 0; Preparative stage
2. Stage 1; Initiation of aseptic cultures
A) Explant
B) Sterilization
C) Browning of medium
•Factors affecting initiation stage
•Conclusions
•References

3. INTRODUCTION
Multiplication of genetically identical copies of a
cultivar by asexual reproduction is called clonal
propagation .
The most significant advantage offered by this aseptic
method of clonal propagation, popularly called
'Micropropagation'- Clonal propagation through
tissue culture.
The size of propagule is small and it produces
‘True-to-type’ products.
It is based on totipotency of the cell. i.e. the ability of
somatic cell of a plant to produce a new complete
plant (defined by F. C. Steward)

4. Advantages of Micropropagation over
the conventional methods
1. In a relatively short time and space a large
number of plants can be produced starting
from a single individual.
2. For orchids, micropropagation is the only
commercially viable method of clonal
propagation

5. HISTORY
1. The history of plant tissue culture starts with the postulates
of cell theory proposed by Schleiden (1938) and Schwann
(1939). According to cell theory:
•Cell is the structural unit of living organism.
•Cells are the functionally independent and complete totipotent
developmental units.
2. G. Haberlandt (1902) first proposed the idea of plant cell
culture under in vitro condition by using an artificial medium.
3. General interest in the use of tissue culture for clonal
propagation of plants originates from the initial success with
orchids, the credit for which goes to G. Morel (1972).

6. Stages of Micropropagation
Micropropagation generally involves four stages:
•Stage 1, initiation of aseptic cultures;
•Stage 2, multiplication;
•Stage 3, rooting of in vitro formed shoots;
•Stage 4, transfer of plants to greenhouse or field conditions
(transplantation).
Murashige (1974) originally described three
basic stages (1-3) for successful micropropagation.
Debergh and Maene (1981) introduced the Stage 0,
making micropropagation a five stage process. Each
stage has its special requirement.

7. 1. Stage 0: Preparative Stage
This stage involves the preparation of mother
plants to provide quality explants for better
establishment of aseptic cultures in Stage 1.
To reduce the contamination problem in Stage I
the mother plants should be grown in a
glasshouse with suitable light, temperature and
growth regulator treatments and drip irrigation
is used.
It would not only help minimize the incidence of
infection in Stage 1 but may also reduce the
need for a harsh sterilization treatment.

8. Examples and advantages of
Preparative Stage
1. Before the introduction of Stage 0 Cordyline cultures could be
initiated only from apical bud; all the nodal segments got infected or
did not respond favorably. With the introduction of Stage 0 both
apical and all nodal segments could be used as primary explants
(Debergh and Read, 1991).
2. Senawi (1985) succeeded in obtaining responding cultures of
Theobroma cacao only from the stock plants grown under glasshouse
conditions.
3. Red light-treated plants of Petunia provided leaf explants which
produced up to three times as many shoots as did the explants from
untreated plants (Read et al., 1978).
4. In woody and bulbous plants suitable temperature treatments should
help in breaking bud dormancy and provide more responsive explants

9. 2. Stage 1: Initiation of
cultures
This stage depends on 3 factors. They are as follows:
A) Explant
B) Sterilization
C) Browning of medium
Initiation and aseptic establishment of pathogen-
eradication and responsive explants is the goal of
this stage.

10. A) Explant
Explant is a tissue taken from mother plant,
cultured under aseptic condition on a defined medium
by manipulation of medium component and other
physiological parameters. The nature of the explants to
be used for in vitro propagation is, to a certain extent,
governed by the method of shoot multiplication to be
adopted.

11. S.No. Explants used Needs
1. Explants with pre-
formed vegetative bud
For enhanced axillary branching.
2. Shoot tips To produce virus-free plant from an infected
individual.
3. Nodal cuttings When the stock plant is virus-tested or,
alternatively, virus eradication is not desired.
4. Runners tips For rhizomatic plants, such as strawberry and
Boston fern.
5. Explants are derived
from root, stem, leaf or
nucellus based on their
natural capacity to form
adventitious buds.
For multiplication through adventitious bud
formation, with or without callusing
6. Leaf-base and scale-
base with small basal
plate.
For monocots.
Table: Type of explants used

12. Meristem-tip culture may also result in the loss of certain
horticultural characters which are controlled by the
presence of virus, such as the clear-vein character of the
geranium. (Cassells et al., 1980).

13. B) Sterilization
Sterilization is very important in order to reduce contamination.
Contamination can be reduced by following steps:
Discarding the surface tissues from plant materials while preparing
the explants minimizes the loss of cultures due to microbial
contamination.
Chances of contamination are much higher in the cultures of
terminal cuttings and whole buds than that in the cultures of 0.5-1
mm shoot tips excised after removing several layers of older leaves.
Shoot tips with sufficient covering of mature leaves, or scales from
the centre of the bulbs, may be dissected out sterile by wiping the
buds or bulbs, respectively, with 70% ethanol and gently peeling off
the outer covering (Hussey, 1980).

14. Fungicides- Bavistin acts on fungal pathogens by inhibiting
the development of germ tubes, the formation of appressoria
and the growth of mycelia. Dithane M45 disrupts the
respiratory activity of the target fungi at several points of
metabolic pathway and is active against spore germination.

15. The most frequently used cytokinins (1-2 mg/L) are
N6- benzyladenine (BA), Kinetin (Kin) and N6 -(2-
isopentenyl)-adenine (2-iP). The naturally occuring
auxin (0.1-1mg/L) indole-3-acetic acid (IAA) is the
least active, whereas the stronger and more stable
compounds α-naphthalene acetic acid (NAA) , a
synthetic auxin and indole-3-acetic acid (IBA), a
naturally occuring auxin, are most common used.

16. C) Browning of medium.
A serious problem with the culture of some plant
species is the oxidation of phenolic substances
leached out from the cut surface of the explant. It turns
the medium dark brown and is often toxic to the
tissues. This problem is especially common with the
adult tissues from woody species. Accumulation of
these phenolic oxidation products can kill the explant.
Procedures used to decrease tissue
browning include the following:
Use of liquid medium with frequent transfer.
Adding antioxidants such as ascorbic acid or
polyvinylpyrrolidone (PVP).
Addition of activated charcoal.
Culture in reduced light or darkness.

17. Factors affecting initiation stage
1. Salt mixture and organic nutrients
• For some plants, the level of salts in the MS medium is either toxic or
unnecessarily high (Anderson, 1975; Adams et al., 1979a). Blueberry
shoots, for example, grow extremely well in a medium with MS salts
reduced to one-quarter strength; higher levels are either toxic or without
any beneficial effect (Cohen and Elliott, 1979).
•
•The promoting effect of diluted mineral salt solution on shoot
proliferation is probably due to reduced nitrogen level. In several
Gymnosperms shoot bud differentiation was promoted by lowering
nitrogen level in the MS medium.
•Organic nutrients of MS or LS media are generally adequate for the
micropropagation of most species.

18. 2. Plant Growth Regulators (PGRs)
The requirement for growth regulators varies with the system and the
mode of shoot multiplication. A higher cytokinin- to-auxin ratio promotes
shoot formation and a higher auxin-to-cytokinin ratio favours root
differentiation.
In some woody species GA3 has been used in the shoot proliferation
medium to improve shoot elongation.
3. Agar content
Since semi-solid cultures are easier to handle and maintain,
the media for micropropagation are traditionally gelled with 0.6-0.8%
agar. However, for several systems liquid medium has proved either
critical for their survival in culture or beneficial for multiplication.
4. pH of medium
Generally the pH of the medium is set at 5.8 . Lowering the pH
to 4, with doubling of Ca+2 and Mg+2 promoted shoot multiplication and
elongation in chestnut (Chevre et al., 1983).

19. 5. Light
Light is required by these cultures for certain
morphogenic processes. A light intensity of 1000-5000 lx is
adequate for this purpose (Hussey, 1980). Optimum light
intensity for shoot multiplication in Gerbera and many other
herbaceous species was reported by Murashige (1974) to be
1000 lx. In low light intensities, the shoots are greener and
taller (Murashige, 1977). Photoperiodism is, of 16 h day
and 8 h night is found satisfactory (Murashige, 1977).
6. Temperature
Cultures are usually maintained at a constant
temperature around 250C However, for tropical species
a higher temperature (300C may be beneficial.

20. Conclusions
From the commercial viewpoint, micropropagation
is the most important aspect of plant tissue culture. Industrial
application of this technique started in the late 1960s and early
1970s, first with orchids and later with other crop plants.
The micropropagation industry is capital and
labour intensive. Rapid multiplication of plants under disease-
free in vitro conditions is often easy. Micropropagation is based
on cellular totipotency.
Initiation stage mainly depends on 3 factors i.e.
the type of explant used, sterilization of medium and explant and
browning of the tissue.

21. References
1. Plant Tissue Culture, a Revised Edition – S. S.
Bhojwani and M. K. Razdan
1990 Elsevier Science B.V.
2. Plant Tissue Culture, Development and
Biotechnology – Robert N. Trigiano, Dennis J. Gray
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