Presented by Under the Supervision of

Md Abul Barkat Dr. Mohd.Mujeeb

M.Pharm. 3rd sem.(2010-11) D/o Pharmacognosy and
Phytochemistry
D/o Pharmacognosy and Phytochemistry Faculty of Pharmacy
Faculty of Pharmacy Jamia Hamdard
Jamia Hamdard
Contents

 Introduction
 Aims and objective
 Plan of work:
• Targets achieved
• Targets to be achieved
• Rationale of the work
 References
Catharanthus roseus var.alba
Introduction

 The plant tissue culture is refers to technique

of growing plant cells, tissues, organs, seeds or
other plant parts in a sterile environment on a
nutrient medium.

 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
Tissue culture offers numerous significant benefits
over traditional propagation methods

 Propagation can be much more rapid than by

traditional means.
 It may be possible in vitro to multiply plants
that are very difficult to propagate by cuttings
or other traditional methods.
 Large numbers of genetically identical clones
may be produced.
 Seeds can be germinated with no risk of
damping off/predation.
 Under certain conditions, plant material can be stored in vitro
for considerable periods of time with little or no maintenance.
 Tissue culture techniques are used for virus eradication,
genetic manipulation, somatic hybridization and other
procedures that benefit propagation, plant improvement, and
basic research.
 Tissue culture is an essential part of many genetic
transformation protocols.
Aims and Objectives

 Selection of suitable culture conditions to initiate, development

and maintenance of callus culture of the proposed plant.
 Development of the biomass contains higher concentration of
secondary metabolites.
 Phytochemical analysis of the developed biomass.
 Optimization of the culture parameters for the enhancement of
the secondary metabolites in cultures.
 Development and evaluation of formulation of the
proposed plant.
 Phytochemical screening to confirm the presence or
absence of phytopharmaceuticals.
 Pharmacological evaluation of callus culture with specific
references to antioxidant and anti-diabetic activity.
Plan of work

Tissue culture studies

 Procurement and identification of plant material.
 Development and optimization of method for sterilization
of explants.
 Micropropagation.
 Preparation of the culture media.
 Initiation, development and maintenance of callus culture.
 Growth kinetic studies
 Optimization of the culture parameters for the enhancement
of the secondary metabolite in callus culture.
 Analytical work

 Phytochemical screening to confirm the presence or absence

of the phytopharmaceuticals.
 Quantification of total phenolic and flavonoid contents.
 HPTLC fingerprinting profile of natural plant parts and their
cultures.
 Quantification of secondary metabolite by the use of HPTLC/
HPLC technique.
Pharmacological work
 Anti-oxidant activity
 Anti-diabetic activity
Targets Achieved

Procurement and identification of plant material

The plant material was collected from the herbal garden of Jamia Hamdard,
and authenticated by Taxonomist, Department of Botany, faculty of Science,
Jamia Hamdard, New Delhi-62
Surface sterilization of explant

S. No Sterilizing agent Conc. (%) Contact time (min.) Observation

1 Sodium hypochlorite 3 10 +
sol.
2 Sodium hypochlorite 4 8 +++
sol.

Observation: 4% Sodium hypochlorite sol.treatment for 7-10 min. showed healthy growth
without contamination.
Preparation of Culture media
 Murashige & Skoog’s (MS) medium was selected as the optimal culture
medium for initiation and development of static culture from the leaf and
seeds of Catharanthus roseus var.alba which was supplemented with the
various combinations of phytohormones for the induction of callus cultures.
Abbreviations used further
 2,4-D = 2,4-Dichlorophenoxyacetic acid
 NAA= Naphthalen acetic acid
 IBA=Indolebutyric acid
 IAA=Indoleacetic acid
 6-BA= Benzyladenine and
 K= Kinetin
 MS + Hormonal Callus initiation Time (days) Observation Figure
Combination
(ppm)

MS + 6-BA +++ 40 Initiation of callus

(1ppm) from leaf

MS +NAA ++ 45 Initiation of callus

(1ppm) from leaf

(–) = No initiatin, (+) = initiation, (++) = Good initiation and (+++) = better initiation
 Targets to be Achieved

 Tissue culture studies

Growth kinetic studies
Development and maintenance of leaf and seed culture.
Optimization of culture parameters for the enhancement of ajmalicine in
callus culture.
Development and evaluation of formulation of the proposed plant.
 Analytical work
Phytochemical screening to confirm the presence/absence of
phytopharmaceuticals.
Quantification of total phenolic and flavonoid contents.
HPTLC fingerprinting profile of natural plant parts and their cultures.
Quantification of ajmalicine in natural plant parts and its cultures.
 Pharmacological work
In vitro antioxidant activity.
Anti-diabetic activity.
Rationale of the work

 The natural source of ajmalicine is very few plant species

like Vinca and Rauwolfia which contain low concentration.
 Due to the high Global demand of this compound, new
methods are needed to enhance its production .
 One of the strategies to enhance metabolite production
using suspension cultured cells in which we use like
elicitors, precursor, adsorbent and immobilization or
biotransformation technique.
 In the present investigation, an attempt will be made to
optimise the culture conditions by L.Almagro, A.J.Lopez
Perez and M.A.Pedreno response to using plant cell culture
based on the use of cyclodextrine for the enhancement of
ajmalicine content in leaf callus of Catharanthus roseus.

 We will also develop novel methods to increase yields of

ajmalicine in the callus of the white vinca . No such type of
systemic tissue culture profile of this plant has been
established by any research worker and agencies.
 References
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 Dahleen, L. S. and Bregitzer, P. (2002). An improved media system

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 Walden, R. and Wingender, R. (1995). Gene-transfer and plant-

regeneration techniques.Trends in Biotechnology, 13, 324–31.