Arora R. (ed.) Medicinal Plant Biotechnology
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x Contributors
Shri Ram
, Department of Bioinformatics and Biotechnology, Jaypee University of
Information Technology, Waknaghat, Solan-173215, Himachal Pradesh, India
Bikram Singh, Natural Plant Products Division, Institute of Himalayan Bioresource
Technology, Council of Scientific and Industrial Research, Palampur-176061, India
Dharam Singh, Lab No. 419, Institute of Molecular Biology, Academia Sinica, Nankang,
Taipei-115 29, Taiwan
Harvinder Singh, Department of Bioinformatics and Biotechnology, Jaypee University of
Information Technology, Waknaghat, Solan-173215, Himachal Pradesh, India
Ajit Varma, Amity Institute of Microbial Technology (AIMT), Amity University Uttar
Pradesh (AUUP), Block 'E-3', IVth Floor, Sector 125, Noida-201303, India
Rajeshwar Verma, Department of Chemistry, Pomona College, 645 N. College Avenue,
Claremont, CA 91711, USA
Heribert Warzecha, Technical University Darmstadt, Institute for Botany,
Schnittspahnstrae 3-5, 64287 Darmstadt, Germany
Christoph Wawrosch, Department of Pharmacognosy, Plant Tissue Culture,
PharmaCenter, Vienna, Althanstr.14, A-1090, Vienna, Austria
xi
Preface
The ancient physicians of India said and proved that ‘there is no plant in this world which
is not a medicine’. Scientists too have begun to realize that if we intend to provide
affordable and accessible healthcare globally, we should sustainably utilize the available
medicinal plant biodiversity. Plant-derived drugs can be a panacea, provided we utilize
them on a scientific basis. Medicinal and aromatic plants (MAPs) are nature’s gift and have
been used for allaying disease and improving the quality of life for ages. The area of
medicinal plants is once again witnessing burgeoning interest in view of their colossal
potential to prevent and cure virtually any disease. MAPs continue to and will remain
invaluable sources of new chemical entities in future.
In recent years compounds derived from MAPs have been commercially exploited by
the pharma, food, flavour, fragrance, dyeing and pesticide industry. The disparate
relationship between demand and supply has necessitated conventional and modern
research in the field. A paradigm shift towards biotechnological applications is evident
since modern biotech tools offer novel opportunities not only for improved production of
useful compounds, but also development of designer plants, a feat impossible to
accomplish by conventional approaches. The future appears bright, but the proof of the
pudding lies in the fact that the designer crops should find commercial application and a
long way would have to be traversed before the lab to land to market concept becomes a
reality.
The present book has been conceived with the intention of providing the state-of-the-
art in the subject and discusses the various facets in the light of contemporary
developments. The book is organized into ten major sections.
Section I highlights the emerging trends in medicinal plant biotechnology, emphasizing
the current state of expertise available at the hands of researchers for production of a
plethora of medicinal compounds in plants cell factories.
Indiscriminate population growth, coupled with urbanization and overharvesting has
led to erosion of precious genetic resources. In addition, extensive human intervention has
resulted in global climate change, which is taking a heavy toll on natural biodiversity.
Further climate change might usher in more vagaries of environment and several medicinal
plant species may become endangered or extinct in coming times. The need of the hour is
to conserve the MAPs and step up efforts to develop improved diverse environment-
tolerant strains. Section II discusses the tools available for conservation of genetic
resources, and the potential and opportunities of microsatellite markers in genetic diversity
screening.
Section III of this book provides a glimpse of the role micropropagation can play in
mass multiplication of elite genotypes of MAPs. The finer nuances of in vitro secondary
metabolite production form the basis of Section IV.
Hairy roots offer immense potential for the production of secondary metabolites as
they are amenable to upscaling in bioreactors. Hairy roots can be utilized for
phytoremediation, and via metabolic engineering it is feasible to overexpress genes and
produce recombinant proteins. Section V deliberates on the role of hairy roots in secondary
metabolite production and discusses the biotechnological applications of plant–microbe
interactions.
Genomic and proteomic approaches have been utilized for understanding the intricate
mechanisms involved in secondary metabolite synthesis. Section VI discusses important
analytical platforms and databases ranging from plant transcriptomics to metabolomics.
A new trend is fast emerging with the hitherto inconceivable amalgamation of
computers and biology/biotechnology. Section VII focuses on the bioinformatics tools for
virtual screening of anticancer drugs. Though still in a stage of infancy, bioinformatic
approaches are going to become the mainstay of medicinal plant biotechnology in future.
Section VIII discusses the potential of molecular tools for characterization of
phytodiversity, while Section IX exclusively focuses on molecular farming for production
of biopharmaceuticals. Transgenic medicinal plants that produce edible HIV, hepatitis,
cholera vaccines and the like will be in great demand in coming years.
Section X deliberates on the use of a multipronged approach in the discipline of
medicinal plant biotechnology so that leads from diverse disciplines like chemistry,
pharmacology and nanotechnology can be integrated.
The hope and hype associated with developments in the field will have to be confronted
head on with a greater degree of maturity in the future.
I must humbly admit that it is impossible to cover all aspects of medicinal plant
biotechnology in a small book like this; nonetheless, an attempt has been made to focus on
some important recent developments and upcoming trends. It is hoped that the readers will
be benefited from this endeavour.
20 October, 2010 Rajesh
Arora
(rajesharoradr@rediffmail.com)
The views expressed here and elsewhere in the book are my own and not those of the
Government of India.
xii
xiii
Foreword
Medicinal plants have been man’s best friend for a long time. Early civilizations relied
exclusively on the medicinal plants for treating their various ailments and slowly what
started as primitive herbal medicine around the world evolved into full-fledged systems
like Ayurveda, Unani, Chinese, European, Japanese and Korean systems of medicine,
primarily focusing on holistic healing encompassing prophylaxis and therapeutics. The
utilization of medicinal plants for a plethora of applications has resulted in renewed interest
for they offer practical and safe medical solutions for use by the masses.
Though earth has been bestowed with a rich biodiversity, overexploitation of medicinal
plants has led many of them to the verge of extinction. On the other hand, medicinal plants
often have to tolerate extreme milieu; they are vulnerable to bacterial/fungal/viral
infections themselves and in order to survive continuously evolve and develop mechanisms
to synthesize secondary metabolites. However, the yield of secondary metabolites in plants
grown in the field is often low at times to be able to meet the burgeoning demand.
It has been realized over the years that plant improvement is essential, especially for
obtaining higher yields of high-value, low-volume medicinally useful compounds.
Conventional genetic approaches have helped in myriads of ways by improving plant
strains and productivity of secondary metabolites. None the less, the need has been felt for
improved procedures and protocols for medicinal plant improvement and it is here that
plant biotechnology has carved a niche for itself.
Medicinal plant biotechnology continues to be an emerging area and besides effecting
improved secondary metabolite production, it has positively impacted conservation of elite
strains. Biotechnological interventions have helped in selection, multiplication,
improvement and better molecular analysis of elite medicinal plants. Genetic
transformation has served as a powerful tool for enhancing the productivity of secondary
metabolites and production of novel medicinal plants. Several new strains/varieties of
medicinal plants have been developed that are highly resistant to diseases, vagaries of
environment, and produce entirely different novel compounds that are not found in nature’s
repertoire, utilizing rDNA technology and taking leads from advances in genomics and
proteomics. Up-scaling of secondary metabolite production in bioreactors has led to
reduction of cost to a great extent, while DNA microarrays are increasingly being utilized
as high throughput screening tools for the simultaneous analysis of multiple genes and
gene expression to gain information on the intricate regulatory molecular pathways. The
newly emerging field of bioinformatics of medicinal plants is another area where there is
immense scope for research. Edible vaccines have already been produced in plants and will
shortly hit the market. In coming years, we might witness several more exclusive designer
medicinal plants with multiple traits which could serve not only as a panacea, but also beat
climate change.
I am extremely happy to learn that Dr Rajesh Arora has envisaged a book on this
important emerging cross-cutting area, covering the latest aspects in the field of Medicinal
Plant Biotechnology, by bringing together eminent scientists from across the globe. I am
sure the book will be useful to researchers, academicians and students alike.
I congratulate Dr Arora on his endeavour and wish the book all success.
Dr W. Selvamurthy
Distinguished Scientist and Chief Controller Research and Development
Defence Research and Development Organization (Government of India), New Delhi,
India
xiv
Acknowledgements
I would like to express my deepest sense of gratitude to the following for providing
inspiration and support in manifold ways:
Dr V.K. Saraswat
Scientific Adviser to Defence Minister
Secretary and Director General Research and Development (DRDO)
Ministry of Defence, Government of India
New Delhi
Dr W. Selvamurthy
Distinguished Scientist and Chief Controller Research and Development (LS)
DRDO Headquarters
New Delhi, India
Dr R.P. Tripathi
Outstanding Scientist and Director, INMAS, Delhi, India
My family has always been a source of inspiration! Mrs Shanti Arora and Mr Gopal
Kumar Arora, my parents, kindled hope in me greatly influencing my thoughts. My wife,
Mrs Preeti Arora, has been a pillar of strength and kept pushing me to complete my targets
whenever I was indolent. Without her support, this book would not have seen the light of
the day. No doubt, I had to ignore my family during this project, but they never minded my
deliberate neglect nor expressed it, rather supported me. I can’t thank them enough! My
bigger family has in no less way contributed to my endeavours. I am thankful to all my
other family members and friends for moral support. My teachers were a constant source of
inspiration and persuaded me to put in my best.
I deem it my duty to sincerely thank all the contributors, who cooperated
wholeheartedly to make this book a success. While I neglected my immediate family, I had
another family and obviously what else could I ask for! It was a wonderful experience
working with this family too!
Another part of my family has been CABI Publishing. They not only set the deadlines,
but also helped me meet it. My sincere thanks go to Ms Rachel Cutts, Associate Editor,
CABI Publishing, Wallingford, Oxon, UK for persuading me to compile this book and
constantly encouraging me. I am extremely grateful to Dr Nigel Farrar, Book Publisher,
CABI, who whole-heartedly supported the publication of this book and became a dear
friend, who not only discussed the book but spiritual issues above the human plane. It has
been my pleasure knowing him in person. I thank Ms Fiona Chippendale nee Harrison,
Production Editor, CABI, for taking care of the finer nuances of publication.
Last, but not the least, I am grateful to Almighty for giving me the requisite strength
and enlightening my intellect to compile this book! Like Mother Teresa used to say, I must
admit that I have only been a pencil in the hands of a writing God!
October, 2010 Rajesh
Arora
xv
The Editor
Dr Rajesh Arora is a senior scientist with India’s Defence Research and Development
Organization (DRDO). His contributions in the area of novel drug design and
development, particularly radiation countermeasure agents of natural origin and
augmentation of medicinally useful secondary metabolites using biotechnological
interventions have received wide acclaim. Dr Arora’s name figures in the Who’s Who of
the World, USA, Who’s Who in Science and Engineering, USA and International
Biography, UK. He is a recipient of several prestigious awards and fellowships. Dr Arora
has been a Visiting Scientist in the European Union during 2009–10. Dr Arora has
published extensively in peer reviewed international journals and has 12 patents and 6
books to his credit. He is a life member of several professional societies and is on the
editorial board of numerous journals. Several of his students occupy responsible positions
in the government/academia/industry. Dr Arora received his PhD in Biotechnology jointly
from Central Institute of Medicinal and Aromatic Plants, Lucknow, and University of
Meerut. He also holds post-graduate diplomas in Human Resource Development and
Marketing Management and an MBA degree in Human Resource Management. Dr Arora
was professionally trained at the Central Drug Research Institute, Lucknow, Sanjay Gandhi
Post-Graduate Institute of Medical Sciences, Lucknow, Defence Institute of Advanced
Technology, Pune, Defence Institute of Physiology and Allied Sciences, Delhi, and
Institute of Technology Management, Mussoorie. Dr Arora also coordinates Technical and
Human Resource Development activities for leveraging human capital in cutting edge areas
of S&T.
He can be contacted via e-mail: rajesharoradr@rediffmail.com
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©CAB International 2010. Medicinal Plant Biotechnology 1
(ed. Rajesh Arora)
Chapter 1
Emerging Trends in Medicinal Plant
Biotechnology
Rajesh Arora, Archana Mathur and Ajay K. Mathur
Introduction
The consumption of herbal medicines is increasing world over, primarily due to the
benefits it offers. As per World Health Organization estimates, nearly 80% of the world’s
population in developing countries relies primarily on herbal medicine for meeting their
healthcare needs (Vines, 2004; Sharma and Arora, 2006; Arora, 2010). Normally,
medicinal plants are harvested from the wild, which causes habitat destruction leading to
depletion of the irreplaceable genetic diversity. Systematic cultivation of medicinal plants
has been proposed and adopted quite often as a substitute to collection from the wild and
has the following advantages: (i) optimization of yield and high quality product; (ii) no
problem of mistaken identity; (iii) reduction of genetic and phenotypic variability; (iv) the
issue of contaminants is taken care of; (v) the problem of variability in extracts and their
instability is reduced; and (vi) availability in large quantities without disturbing the natural
environment. However, sometimes the spectra of compounds produced by medicinal and
aromatic plants (MAPs) under cultivation are quite different from those of wild
populations. In order to obtain elite germplasm with enhanced qualities conventional plant-
breeding methods have been extensively employed to improve agronomical as well as
medicinal traits and molecular marker assisted breeding has been used with substantial
returns on investment. None the less, it has been felt that significant improvement of MAPs
for meeting the ever increasing demand of the industry in future is possible only by
employing biotechnological interventions.
Phytomedicines Drive the Biotech Industry
MAPs have been utilized for the prevention and treatment of diseases for a long time. In
recent years, a plethora of drugs have been derived from medicinal plants e.g., reserpine, an
antihypertensive alkaloid from Rauvolfia serpentina, vinblastine, an antitumour alkaloid
from Catharanthus roseus, podophyllotoxin from Podophyllum hexandrum and P.
2 Emerging Trends in Medicinal Plant Biotechnology
peltatum, morphine, a narcotic derived from Papaver somniferum, cocaine, a local
anaesthetic and a potent CNS stimulant from Erythroxylon coca, strychnine, a nerve
stimulant from Strychnos nux vomica, caffeine from tea, coffee beans and cocoa, quinine
from Cinchona ledgeriana and C. succirubra, artemisinin, an antimalarial drug from
Artemisia annua, atropine, a parasympatholytic agent from Atropa belladonna,
camptothecin, the chemotherapy drug from Camptotheca acuminata, taxol, the anticancer
drug from Taxus brevifolia and Taxus baccata, papaverine from Papaver somniferum,
silybin from Silybum marianum, hyperoside from Crateaegus laevigata, anticancer
genistein and diadzein from Glycine max, hypericin from Hypericum perforatum etc.
(Sharma and Arora, 2006; Arora, 2010). MAPs are not only important as drugs, but are a
repository of future drugs that can solve emerging diseases e.g., AIDS, novel H1N1 flu
(Arora et al., 2011) and emerging problems of modernization like chemical, biological,
radiological and nuclear (CBRN) threats (Arora, 2008).
Natural plant products have served as important sources of raw materials for food,
pharma, cosmetics and nutraceutical industries (Phillipson, 2003; Oksman-Caldentey and
Inze, 2004). The medicinal importance of MAPs has necessitated research and this in turn
is the driving force for the biotech industry.
Conservation of Medicinal Plants: the Crucial Role of Biotechnology
Natural selection over the course of years has led to the evolution of MAPs with diverse
traits. This genetic diversity has been harvested for human use per se and also forms the
basis for selection of new and improved varieties of MAPs, which researchers utilize for
development of new chemical entities with diverse medical applications. There has been a
rapid erosion of the important natural resource of medicinal and aromatic plants due to a
variety of reasons including: (i) overharvesting from the wild for commercial use since it is
cheaper; (ii) rapid and extensive population growth leading to irreparable environmental
damage; (iii) extensive industrialization; (iv) deforestation; (v) substitution of local
genotypes with improved varieties and hybrids; (vi) forest fires; (vii) natural disasters;
(viii) climate change; (ix) development of land for agriculture; (x) changes in agricultural
practices; and (xi) abuse of pesticides, weedicides and other agrochemicals etc.
Consequently, a rapid and profound erosion of invaluable bioresource has occurred, often
leading to loss of several species whose true potential had hardly been explored.
The herbal industry has paid scant attention so far to the efficient utilization and
conservation of natural resource in the environment. It is, therefore, imperative to conserve
this precious bioresource. Both in situ and ex situ methods of conservation have been
utilized in recent years and are essential. However, it has been unanimously felt that
biotechnological interventions are essential for selection, maintenance and multiplication of
the elite genotypes of MAPs.
Tissue culture has been widely employed for conservation of MAPs. However, tissue
culture-based approaches aimed at in vitro conservation of MAPs should be able to
efficiently and rapidly multiply and preserve genetic stability of the plant material.
Conservation of germplasm using tissue culture techniques can be envisaged either as
short- and medium-term conservation or as long-term conservation/cryopreservation.
Molecular techniques are increasingly being employed for germplasm characterization.
However, a lot needs to be done in this area. The coming years are likely to witness more
Emerging Trends in Medicinal Plant Biotechnology 3
efforts in the direction of conservation of medicinal and aromatic plants since climate
change is taking a heavy toll and several useful plant species are on the verge of extinction.
Chapters 3 and 4 of this book discuss some of the issues relating to ex situ and in situ
conservation of Podophyllum hexandrum, an important medicinal plant species, of the
Himalayan region.
Micropropagation (Quality Assurance and Quality Control)
Plant tissue culture techniques offer a viable solution for the production of standardized
quality phytopharmaceuticals through mass-production of consistent plant material for
physiological characterization and analysis of active ingredients. Micropropagation
protocols for cloning a variety of MAPs have been developed over the years (Rout et al.,
2000; Nalawade and Tsay, 2004; Rathore et al., 2010). Integrated approaches of
micropropagation are needed to provide a basis for the development of novel, safe,
effective, and high-quality commercial products.
Micropropagation can be utilized for rapid multiplication of elite clones. The
advantages offered by micropropagation of medicinal plants are many and are discussed in
Chapter 6. Although micropropagation is a multi-billion dollar industry, the share of MAPs
as of now is miniscule, though efficient protocols for several MAPs are available. It is
anticipated that with the realization of the importance of quality medicinal plants for the
pharmaceutical industry and rapid depletion of natural populations, more attention will be
paid to this area. Image-guided robotics for automation of micropropagation will help
reduce costs, while assuring quality assurance and quality control.
Micropropagation by conventional techniques is a labour-intensive method.
Automation of micropropagation in bioreactors has been suggested as an economically
viable solution (Cazzulino et al., 1991). Photoautotrophic micropropagation systems
(entirely dependent on photosynthesis of plants in vitro) usually result in improved growth,
lower loss due to contamination, better quality, elevated percentage survival ex vitro and
lower production costs and have been advocated for commercial production of
micropropagated plantlets of medicinal plant species.
Cell and Tissue Cultures: Do They Represent Factories of the Future?
Secondary metabolites are often produced in miniscule quantities in the plant and the ever
increasing demands of the industry can hardly be met through this approach. Therefore,
attention has been paid to development of biotechnological methods. Plant cell and tissue
cultures represent a viable renewable resource of industrially important plant natural
products, required for food, drug, fragrance, flavour and dye industries (Phillipson, 2003;
Mulabagal and Tsay, 2004; Oksman-Caldentey and Inze, 2004). Some of the exclusive
advantages that cell and tissue culture-based production technology provide include the
following: (i) control of product supply independent of the geographical availability of
plant; (ii) no constraint of seasonal fluctuations; (iii) uniform growth under defined
environment; (iv) shorter life cycle free of seasonal or batch-to-batch variation; (v) bio-
transformation through feeding and elicitation is possible; (vi) simpler organization
provides minimum structural constraints and neighbouring cell interference; (vii) ease with