This document describes the process of plant tissue culture using Coleus forskohlii as an example. Key steps include collecting and sterilizing explants from the plant, culturing the explants on Murashige and Skoog medium, and subculturing to induce shoot formation using different concentrations and combinations of plant growth regulators. Initial results showed shoot initiation from shoot tip explants on medium with 2.0 mg/L BAP, callus formation from leaf explants on medium with 1.0 mg/L Kn and 2.0 mg/L BAP, and multiple shoot formation on medium with 1.5 mg/L BAP. The goal is to develop a mass propagation protocol for this plant through

1. Plant Tissue Culture
Raj Kamal Vibhuti
Department of Microbiology

2. “Tissue culture is the term used for “the
process of growing cells artificially in the
laboratory”
Tissue culture produces clones, in which all product
cells have the same genotype (unless affected by
mutation during culture)
Multiple plant produces in short time with the help of
Plant tissue culture.

3. Plant tissue culture had its origins at the beginning of
the 20th century with the work of ‘Gottleib Haberlandt’.
History

4. I. The first commercial use of plant clonal
propagation on artificial media was in the
germination and growth of orchid plants, in the
1920’s.
II. In the 1950’s and 60’s there was a great deal of
research, but it was only after the development of
a reliable artificial medium (Murashige & Skoog,
1962) that plant tissue culture really ‘took off’
commercially.
III. A more recent advance is the use of plant and
animal tissue culture along with genetic
modification using viral and bacterial vectors and
gene guns to create genetically engineered
organisms.

5. What We Need For Plant Tissue Culture?
Appropriate tissue / Explants
A suitable growth medium containing energy sources and
inorganic salts to supply cell growth needs. This can be liquid or
semisolid media.
Aseptic (sterile) conditions, as microorganisms grow much more
quickly than plant and animal tissue and can over run a culture.
Growth regulators - auxins & cytokinins (Mainly)
Frequent subculturing to ensure adequate nutrition and to
avoid the build up of waste metabolites.

6. Plant Tissue Culture Media
“A nutritive solution, solid or liquid, for
culturing cells.”
Functions of culture medium---
I. Provide water
II. Provide mineral nutrition
III. Provide vitamins
IV. Provide growth regulators
V. Access to atmosphere for gas
exchange
VI. Removal of plant metabolite
waste

7. Important Plant Tissue Culture Media

8. Culture Medium

9. Murashige & Skoog(MS) Media

10. Plant Tissue Culture / Micropropagation
1. Selection of the plant tissue (explant) from a healthy vigorous
‘mother plant’.
2. Establishment of the explant in a culture medium. The
medium sustains the plant cells and encourages cell division.
It can be solid or liquid. Each plant species (and sometimes
the variety within a species) has particular medium
requirements that must be established by trial and error
method.
3. Multiplication- The explant gives rise to a callus, which is
manipulated by varying sugar concentrations and the auxin
(low): cytokinin (high) ratios to form multiple shoots.
4. Root formation - The shoots are transferred to a growth
medium with relatively higher auxin: cytokinin ratios
5. Hardening- The rooted shoots are potted up (deflasked) and
‘hardened off’ by gradually decreasing the humidity.After that
new plant transferred to field.

12. Why do Plant Tissue Culture?
A single explant can be multiplied into several
thousand plants in less than a year.
Taking an explant does not usually destroy the
mother plant, so rare and endangered plants can be
cloned safely. Once established, a plant tissue culture
line can give a continuous supply of young plants
throughout the year.
Meristem tissue culture to provide virus free plants.
Tissue culture allows fast selection for crop
improvement - explants are chosen from superior
plants, then cloned.
Tissue culture clones are ‘true to type’ as compared
with seedlings, which show greater variability.

13. Advantages of plant tissue culture
1. The biochemical engineer can grow plant cells in liquid culture on a
large scale—Bioreactor
2. The production of dihaploid plants from haploid cultures shortens the
time taken to achieve uniform homozygous lines and varieties
3.The crossing of distantly related species by protoplast isolation and
somatic fusion increases the possibility for the transfer and expression of
novel variation in domestic crops
4.Cell selection increases the potential number of individuals in a
screening program.
5.Micropropagation using meristem and shoot culture techniques allows
the production of large numbers of uniform individuals of species from
limited starting material.
6.Genetic transformation of cells enables very specific information to be
introduced into single cells which can then be regenerated.

14. Organogenesis--Shoots or roots are
induced to differentiate from a cell or
cell clusters.
Organogenesis

15. Types of Plant Tissue Cultures
1.Callus Culture
2.Root Culture
3.Apical Meristem Culture
4.Anther Culture
5.Pollen Culture
6.Ovary Culture
7.Embryo Culture
8.Protoplast Culture

16. Protoplast Culture
1.Selection of Explants
2.Isolation of protoplasts
3.Purification of protoplasts
4.Determination of density & Viability
5.Culture of protoplasts
6.Regeneration of plant from protoplast

17. Isolation of protoplasts

18. Purification of protoplasts

19. “Protocol Development for mass propagation of
Coleus forskohlii”
Collection of Plant Material
Coleus forskohlii Briq. (Var K8) is the plant material used for the
present study from were excised as explants from the hardening
unit of Renaissance Biotech Pvt. Ltd. at Khagaul (Patna). Explants
of leaf, node and shoot tip were excised.
Surface Sterilization
Materials:
Explants (leaf, node and shoot tip), Labolin, Bavistin (1%), Mercuric
Chloride (0.1%), Streptomycin (0.5%).

20. Method:
The explants were washed with running tap water to
remove adhering dust particles.
The explants were then surface sterilized with 1%
bavistin and 0.1% mercuric chloride solution for 1-2
minutes followed by 4-5 washing with sterilized
distilled water.
At the end, they were treated with 0.5%
streptomycin solution for 5-7 min followed by washing
with sterilized distilled water.

21. Media and Culture Condition
Materials :
Murashige and Skoog’s (MS) media and Culture Room
(maintained at 22±2ºC)
Method:
After surface sterilization, sterile explants were inoculated on
sterilized solid Murashige and Skoog’s (MS) media with 3%
sucrose.
All cultures were maintained at 22±2ºC and 16 hour
photoperiod (light intensity of 2,000 Lux) at the level of culture
tubes, using white fluorescent lamps.

22. Sub Culture
Explants and calli were sub cultured every 4-5 weeks
interval. The tube containing culture materials were
externally sterilized with 70% alcohol. The materials
were transferred to culture tubes containing fresh
medium with the help of sterilize forceps in the
inoculation chamber. After subculture they were
transferred to the culture room.
Materials:
Murashige and Skoog’s Media supplemented with
Auxin (NAA, 2,4-D) and Cytokinin (BAP and Kinetin)

23. Method:
To test the effect of different growth regulators on
shoot multiplication, mainly two types of cytokinin
were taken.
The Murashige and Skoog’s medium was
supplemented with different concentration (1.0, 1.5
and 2.0 mg/L) of 6-benzylaminopurine (BAP) and
Kinetin (Kn).
Explants and calli were sub cultured every 4-5 week
interval.
After subculture they were transferred to the culture
room.
Regular observation at an interval of two days was
made for the formation of callus, change of colour and
initiation of the root or shoot.

24. Results
Fig 1. Initiation of shoot from the shoot tip explants in 2.0 mg/L BAP

25. Fig -Initiation of callus from the leaf explants in 1.0 mg/L Kn and 2.0 mg/L BAP

26. Fig - Generation of multiple shoots in BAP 1.5 mg/L

27. Fig - White and friable Callus in BAP 2mg/L+2,4-D 1.5mg/L
Fig- Shoot tip explant shows both multiple shooting and callusing in BAP 2.0 mg/L