Journal of Experimental Biology and Agricultural Sciences, August - 2014; Volume – 2(4)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

THE ROLE OF BIOTECHNOLOGY IN THE CONSERVATION OF

BIODIVERSITY

Malabika Roy Pathak* and Mohammad S Abido

College of Graduate Studies, Desert and Arid Zone Sciences Program, Arabian Gulf University, Kingdom of Bahrain

Received – May 19, 2014; Revision – June 07, 2014, Accepted – August 16, 2014
Available Online – August 21, 2014

KEYWORDS
ABSTRACT
Biodiversity
Biological diversity provides the variety of life on the Earth and can be defined as the variability among
and between the living organisms and species of surrounding ecosystems and ecological complexes of
Biotechnology
their life support. It has been estimated that one third of the global plant species are threatened in
Endangered plants different level according to the International Union of Conservation of Nature (IUCN).The major threat
to rapid loss and extinction of genetic diversity due to habitat destruction, pollution, climate change,
Ex situ invasion of exotic species, human population pressure, ever increasing agricultural pressure and
practices, life style change etc. are well-known. Biodiversity conservation is a global concern. All
Genetic diversity member states of the Convention on Biological Diversity (CBD) took measure to preserve both native
and agricultural biodiversity. The global concern of biodiversity conservation initiated either by in situ
In situ or ex situ methods. In situ methods protect both plants and their natural habitat. On the other hand, ex
situ methods involves preservation and maintenance of plant species or plant parts (such as seeds,
Plant Tissue Culture cuttings, rhizomes, tubers etc.) outside their natural habitat for the purpose of developing seed banks or
more preciously gene banks following classical / advanced methods of plant propagation. Classical
In Vitro Gene Bank methods of plant propagations have certain limitations in terms of rapid production of plants or plant
propagules and their long term conservation. So, the biotechnological methods such as plant tissue
culture, plant cell culture, anther culture, embryo culture etc. are quite applicable and useful techniques
for ex situ conservation. On the other hand, the production of superior quality seeds has enhanced by the
application of plant biotechnology. So, plant biotechnology offers new means of improving biodiversity
conservation rather than threatening biodiversity in various ways.

* Corresponding author
E-mail: malabikarp@agu.edu.bh (Malabika Roy Pathak)

Peer review under responsibility of Journal of Experimental Biology and

Agricultural Sciences.

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Plant conservation following natural and semi natural ways either in ex situ or in situ or by both
conservation techniques have applied. The ex situ conservation of wild, rare and endangered plant
species through in vitro plant tissue culture techniques are very useful for conserving biodiversity.
This review highlights how different bio-techniques are useful in conservation of biodiversity in
different ways.
1 Introduction
Biodiversity is the mass of different living beings in a
particular ecosystem or on the whole earth. It exists in three
different levels; genes, species, and ecosystems. Each of the
components has its own composition, structure and function
(Redford & Richter, 2001; Noss, 2005). Biodiversity provides
the basis for ecosystems and their services, upon which all
people fundamentally depended (Cardinale et al., 2012).
Biodiversity is considered as the base of agriculture, source of
all recent crops and domestic livestock species since the
beginning of human civilization. Similarly, the origin of
biotechnology is very deep rooted in the human history from
the starting of domestication of wild plants and animals to
recent time. Genetic manipulation by classical methods of
plant breeding and selection of superior and new varieties
started since prehistoric time. Similarly, biotechnology has
been used to improve and enhance crop productivity, as well as
to conserve, evaluate and utilize the various aspects of
biodiversity (Brink et al., 1999; Wolfe, 2000).
The major causes of loss of biodiversity is due to human
activities such as habitat destruction, pollution, climate change,
invasion of exotic species, human population pressure,
agricultural practices, life style change etc. (Opdam &
Wascher 2004). The United Nations Food and Agricultural
Organization (FAO) assuming that global population is
approaching towards 9.1 billion in 2050 and there is a need of
70% increase in food production (Godfray et al., 2010). In this
current situation, undertaking of effective as well as productive
agricultural land uses has raised a global challenge of
conserving biodiversity (Tscharntke et al., 2012). At the same
time, FAO advised to conserve plant genetic biodiversity as it
is essential for future food security. As the reduction in crop
diversity is causing a potential threat to food security in global
food supplies is an important issue (Khourya et al., 2014).
Hence, the International Treaty on Plant Genetic Resources for
Food and Agriculture came into exist in 2001 to recognize
farmers contribution to the diversity of crops, establish
mechanism to access plant genetic materials and share benefits
of developing genetic materials (FAO, 2014).
It has been estimated that one third of the global plant species
are threatened in different level according to International
Union of Conservation of Nature (IUCN, 2013). The
increasing awareness and great concern about global
biodiversity conservation, the United Nations (UN) declared
the current decade (2011-2020) as the “Decade of
Biodiversity’ and has set 2020 as the target for restoring at
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Pathak and Abido
least 15% of degraded ecosystems as well as conserving 17
and 10% of terrestrial and inland water and marine and coastal
areas respectively (Tscharntke et al., 2012; SCBD, 2014).
Global concern about the loss of valuable genetic resources
stimulated many new programs for the conservation, protection
and management of natural resources and wildlife.
Conservation of biodiversity means protection of valuable
natural resources for future generations as well as well being of
eco-system function. Within past few decades, several
conservation strategies have been developed mainly in the
methods of in situ and ex situ conservation policy.
Biodiversity conservation is based mainly on in situ
conservation where habitats, species and ecosystems are
naturally present and preserved in natural condition without
any changes. Genetic variation is necessary for long term
survival of plant species in natural habitat (Lande, 1988).The
genetic diversity can be maintained by introducing new
individuals to increases population size to minimize inbreeding
depression, genetic drift and extinction risk (Nybom,
2004).The gradual declining of plant species is the common
phenomena of in situ preservation method, due to natural
habitat loss, fragmentation, modification even in reserve area
with time (Fahrig, 1997).
While, ex situ conservation involves preservation and
maintenance of living samples outside their natural habitat
using several techniques such as in botanical gardens, seed
banks, gene banks etc. and is able to solve some problems
related to in situ preservation techniques. Different modified
techniques have applied to preserve different categories of
plant species by using both in situand ex situmethods of
conservation as they are complementary to each other (Khan
et al. 2012). The maintenance and analysis of genetic diversity
are important factors in the preservation of rare and
endangered plant species either by in situ or ex situ methods.
The conservation of plants, seed, pollen, vegetative propagules,
tissue or cell by using plant tissue culture techniques, which is
highly accepted biotechnological approaches for
conservationof rare and endangered plant species (Paunescu,
2009). Moreover, various bio-techniques are not only offer the
possibilities of faster multiplication of clones of endangered
plant species for conservation of genotypes, but also conserve
genetic material, provide the power of modification at genetic
level by changing their expression level
(Verpoorte&Memelink, 2002). Biotechnological methods are
reliable and can provide continuously safe, higher quality
natural products for food, pharmaceuticals and cosmetic
industries, similarly, they are applicable in preserving
biodiversity in several ways (Nalawade et al., 2003; Julsing et
The role of biotechnology in the conservation of biodiversity
al., 2007). Several biotechnological approaches are important
factors to conserve, analyze and detect genetic diversity of rare
and endangered plants such as different molecular marker
techniques starting from biochemical, physiological and DNA
based markers (Khan et al., 2012 ). The present reviewwill
discuss the global impact of biodiversity, factors of
biodiversity loss and use of biotechnology to conserve,
maintain and enhance biodiversity.
2 Global Impact of Biodiversity
Biodiversity is needed for human survival either in direct or
indirect way. The direct use includes things like food, fibers,
medicines and biological use, while the indirect uses include
ecosystem services such as atmospheric regulation, water and
nutrient cycling and industrial raw materials etc (Fahrig 1997;
Redford & Richter 2001). Biodiversity plays effective role in
providing various genetic resources of agriculture which is
important for the biological basis for world food security and
support of human livings. Preservation of genetic resources of
plants of different categories is central not only for global
economy but also to maintain ecological balance. In the field
of agriculture, the genetic diversity of each type of crop
(varieties, cultivars etc.) has considerable importance in crop
improvement programs. Various programs pay attention to
conserve biodiversity for food and agriculture at national and
international levels (Campebell et al., 2010). The germplasm of
a large number of primitive and wild cultivars of several plants
constitute a pool of genetic diversity to support future crop
improvement programs (UNEP 2008). A wide range of genetic
diversity can be introduced in plants either by classical or
advanced technology against the major threats of plant survival
such as disease, pests,environmental stresses etc. ( Cadotte et
al., 2008; Corlett & Primack, 2008).
The biodiversity of organisms has great impact on the
functioning of the natural ecosystem services and ecological
processes for the total function of humankind. Ecosystem
services provide benefits in different ways those are involved
in the production of renewable resources such as food, wood,
increased carbon sequestration, photosynthesis, recycling of
nutrients, air and water purification, pollination, prevention of
soil erosion etc, and regulating services are those that minimize
environmental changes such as climatic change, controlling
pest/disease (Tscharntka et al., 2012). Different ecological
services have significant contribution in the world’s economy
both in industrial and agricultural development either directly
or indirectly. Several industrial products such as oil, rubber,
fiber, building material, timber, wood, paper etc. are widely
obtained from biological resources. Being a member of
ecosystem, microorganisms play a dynamic role in food
processing mechanism in food industry (Cardinale et al.,
2012).
Biodiversity plays a significant role in human health as plants
are great source of medicines, especially traditional medicines,
which are useful in the treatment of various diseases are well
recognized (Alves & Rosa, 2007). Traditional Chinese
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354
medicine, Indian ayurveda and Arabic unani and
ethanomedicine contain thousands of compounds isolated from
leaves, herbs, roots, bark etc. Except these traditional
medicines, large number recent days advanced technology
based pharmaceutical products are also obtained from plants.
Both developing and developed countries are isolating
different drugs following either classical or advanced ways.
Almost 80% of the world population depends on medicines
those are obtained from natural resources either in direct or
indirect ways (WHO, 2002). To add on, nearly 25% of all
commercial medical drugs in developed countries are based on
plants and plant derivatives. This dependency may reach as
much as 75 per cent in case of developing countries (Principe,
1991).
For instance, about 200–250 herbal species used for treating
human illnesses are traded in the Mediterranean region (Lev &
Amar, 2002; Said et al., 2002). The greater diversity of life
increasing the possibility of discovering new medicines and
fostering economic development, as almost every plant species
are of potential use of some commercial or medical value
(Opdam & Wascher, 2004). According to this view, it is
therefore very important to conserve all living species.
Biodiversity not only supply essential materials for human
requirements, it also provide non-material benefits like
spiritual inspiration and visual values, thoughtful artistic
mentality, cultural diversity, aesthetic and natural bonding to
develop, nourish, enjoy, cultivate and express the internal
quality of human being. The beauty inherent in biodiversity is
a great source of pleasure. Although this aesthetic value is
impossible to quantify, but it remains as source of fundamental
development of human being (Sarkar & Frank, 2012).
3 Degradation of Biodiversity
The Earth's environment is changing on all scales from local to
global, in a large measure due to human activities. The rapid
increase in human populations and their increasing needs has
caused a major threat to biodiversity and wildlife in recent
time. In the last 50 years, there is a significant shift in ever
increasing population pressure on earth, life style change,
urbanization, extensive agriculture, overexploitation and
unsustainable utilization of natural resources and important
plant species, introduction of alien species resulted in
unprecedented decline in biodiversity and ecosystem services
(Fahrig, 1997). The most important causes of biodiversity
decline are habitat loss, fragmentation and habitat isolation
(Millennium Ecosystem Assessment, 2005). For instance, the
Mesopotamian marshes of Iraq lost more than 90% of their
original area due to water diversion projects during the 1990s
(SCBD, 2010). The increasing rate of manmade activities
augmented greenhouse gasses and working as key factor in
current global worming around 3-5°C in the next 100 years has
predicted by using several computer models (Giam et al.,
2010). The relation of global warming and climate change and
loss of biodiversity have indivisibly linked with associated
ecosystem services, still there is no ecosystem management
plan has been devised that can assist living organisms in
355
mitigating and adapting to climate change (Schroth &
McNeely, 2011). Climate change and global warming affect
the habitat change of plants, animals, microorganisms and at
the same time affect species at cellular level that may lead to
the alteration at genetic makeup of individual cell. Moreover,
the issue of land use and climate changes has great impact on
worldwide loss of biodiversity (Corlett & Primack, 2008).
Several studies suggest extensive and rapid land use due to
human activities and climate change are the main indicators of
increasing species extinction from natural habitat. Climate
changes affecting plant physiological processes which can
cause shrinking of plant species area wise locally then globally
as a whole. The rate of species loss in recent decades is 100-
1,000 times faster than the natural rate and 60% of the
ecosystems products and services on which we as a society
rely are in degraded or in decline condition (SCBD, 2014).
4 Conservation of Biodiversity
The concept of conservation biology matured in the mid-20th
century as ecologists, naturalists, and other scientists worked
together to address the declining of global biodiversity as the
major issue. The conservation of biodiversity was recognized
in multiple levels of interactions between the international
communities, those led the United Nations to process and
action in the development of the convention of biological
diversity (CBD, 1992). The international understanding
developed to adopt and recognize the conservation of
biodiversity as a global issue and legal binding of sustainable
use of biological resources. The conservation ethic promote the
management of natural resources for the purpose of sustaining
biodiversity in species, ecosystems, evolutionary process,
human culture and society (Soule, 1985). However,
conservation biology reformed around strategic plans with time
to protect regional biodiversity with specific issues in the later
time (Margules & Pressey, 2000). At the same time, priority
has given to the strategic conservation plans to uses public
policy in local, regional and global scales of communities,
ecosystems, and cultures (Gascon et al., 2007). Action plans
identified the ways of sustaining human well-being,
employing natural capital, market capital, and ecosystem
services for the survival of mankind as in recent years, while
increasing loss of biodiversity has posed a serious threat to the
survival of human being (Corlett & Primack, 2008).
5 Techniques for Plant Conservation
Different level of genetic diversity helps plant species to
survive in their natural habitat, so conservation of plants in
their natural habitat play important role. According to the
guidelines of International Union for Conservation of Nature,
both in situ and ex situ methods are well known and applied for
the conservation of Red listed plants (IUCN, 2013). The
choices of one or the other technique, or a combination of both,
depend on the particular case and both are complement to each
other based on the adoption of conservation policy. In situ
conservation method is the most valuable method, as it is
natural and totally depends on the plants in their living forms
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Pathak and Abido
in their natural habitat and allowing natural evolution
(Ashmore et al., 2011).So, in situ conservation involves the
maintenance and protection of natural habitats, while ex situ
conservation involves the conservation or propagation of a
species variety, clone or genetic material of plant species either
in botanical garden, or in the process of seed banks or using
some semi-natural habitat environment. Being a naturally
adopted process of conservation, in situ conservation faced
several problems due to restricted and fragmented habitat,
climate change, unsustainable use of plant resources, attack of
pathogenic organisms and invasive species in natural
environment while ex situ method is more effective and more
scientific because of its way of conservation which is
governable with rules, regulation and adopted methodology in
artificial way ( Al-Eisawi 2003; Reed et al., 2011). Although it
hampers plant evolution, the principle of any applied
technology for germplasm conservation should have to
preserve the maximum possible genetic diversity of a
particular plant or genetic stock for future use. Environmental
factors are the cause for genetic diversity in a natural habitat,
in that case, in situ is the best alternative. Diversity in plants
exists at generic, specific and individuals while specific genetic
stocks can be develop and maintain through breeding,
mutation, plant tissue culture and applying transgenic
techniques to fulfill specific objectives through ex situ
conservation methods (Brink et al., 1999). In situ
conservation maintains and manages existing genetic diversity
and viable populations of wild taxa in order to maintain
biological interactions, ecological processes and functions
under natural conditions (Ashmore, 1997). The ex situ method
follows the propagation of plant species, varieties, specific
clones either by classical method using seed, cuttings,
rhizomes, corms or by using biotechnological methods such
cell, tissue, organ culture, micrpropagation techniques,
cryopreservation, germplasm banking, gene banking, applied
advanced research to introduce new genetic modification in the
existing population, reinforcement of existing population and
reintroduction into the wild controlled environments
(Schemske et al., 1994). The main objective of germplasm
conservation is to maintain constant preservation of germplasm
as it can be available at any time. In nature, propagation by
seed germination is usual, but sometimes seeds fail to
germinate due or incomplete and rudimentary embryos or
environmental reasons (Hamrick & Godt, 1989). In general,
most of the orthodox seeds of different plant species can be
dehydrated down to low water content and thus can be stored
at low temperature for long time without the loss of seed
viability (Dussert et al., 1997). There are abundant forest tree
species, especially tropical forest tress, producing recalcitrant
seeds; those seeds cannot be dried to sufficiently low moisture
level to allow their storage at low temperatures (Roberts,
1973). Moreover, there are large number of plants produce
very few rudimentary and incomplete, heterozygous seeds and
their conservation in seed forms are problematic, those are
categorized as intermediate plants (Ellis, 1991). Conservation
of these plant species by traditional ex situ procedure has
several drawbacks, which limit its efficacy and threaten the
safety of the growing plants genetic resources in the field.
The role of biotechnology in the conservation of biodiversity
Moreover, conservation of rare and endangered plant species
due to their high rate of threats in natural environment need to
be conserved following advanced biotechnological methods of
ex situ conservation process was highlighted after the world
biodiversity declined in unprecedented rate. These
conventional methods of seed and clonal propagation of plant
genetic resources in ex situ conservation process was replaced
by in vitro cultural methods which are more promising and
advantageous of regenerating large numbers of plant
propagules using minimum of start-up plant material (Sarasan
et al., 2006; Rowntree, 2006).
5.1 Biotechnology
Biotechnology has been defined as any technique that uses
living organisms, or substances to make or modify a product,
to improve plants or animals, or to develop microorganisms for
specific uses. It consists of a gradient of technologies, ranging
from the long-established and widely used techniques of
traditional biotechnology to novel and advanced biotechniques
of cell and tissue-culture methods and transgenic methodology.
The beneficial impact of plant biotechnology on improvement
and production of economically important crops are well
established, while it has great impact in conservation of
biodiversity too. Advances in biotechnology, especially in vitro
culture and molecular biology especially transgenic technology
lead to the production of a new category of germplasms, cell
lines with special attributes and genetically transformed
material (Engelmann, 1991). In vitro culture and collection of
germplasms by rapid, medium and slow growing
multiplication processes, slow growth storage,
cryopreservation have great applicability to reduce the risk of
loss of plant genetic resources those are venerable in general
growing and storage conditions (Pence et al., 2002). The huge
loss and degradation of plant genetic resources were partially
assessed, conserved and managed by adopting advanced ex situ
techniques of advanced biotechnological methods especially in
vitro culture and several techniques of molecular biology for
study and analysis purpose of genetic diversity (Ashmore,
1997; Sarasan et al.,2006; Paunescu, 2009).
5.2 Plant Tissue culture
Plant tissue culture (PTC) is a quick, season independent and
efficient in vitro technique to propagate plants under sterile
micro environment. It is very effective method of cloning of
plant material and to develop disease free clean plant stock.
PTC is a sun-rise technology and working as a catalyst of
agricultural and industrial development. Any plant cell has the
power of cellular totipotency to be differentiated into whole
plant in the process of plant tissue culture methodology. There
are different types of culture methods using different organs
(Chawla, 2009). The technique of different types of culture is
applied with several objectives, the most important one is the
enhancement of plant production rate by quick regeneration of
plants in the absence of seed, or otherwise by using the seeds
which have very low chances of germination capability (Ellis,
1991; Abo El-Nil 1997). Different techniques in PTC may
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356
offer certain advantages over traditional methods of
propagation for assembly, proliferation, preservation and
storage of plant genetic resources (Bunn et al., 2007). The
success of plant tissue culture depends on the success of shoot
regeneration in a rapid and reproducible way. It has great
importance in the crop improvement program which is facing
the increasing depletion of natural resources. Moreover, tissue
culture techniques can be applied in germplasm conservation
of medicinally important plants those are of important source
pharmaceutical compounds. It can be applied for regenerating
different clean disease free stock of plants in the field of
agriculture, horticulture, floriculture and pharmaceutical
industry (Fischer et al., 2004). Tissue culture is a useful
technique to preserve somatic embryos which can be applied
in the medium and long-term conservation process. In the
medium–term conservation, there is a need to lengthen the
period between subcultures by reducing growth rate. PTC is
also a great source of creating variations through the
development, selection and isolation somaclones commonly
known as somaclonal variations. Thus biotechnology lead to
the production of a new category of germplasms, clones of
special category, elite cell lines and genetically transformed
material with desired traits. The cultivation and conservation of
the new germplasms in the changed environmental situation
can be able to add some specific impact in changed
environmental situations.
Rapid and mass propagation of plant species and their long-
term germplasm storage can be achieved in a small space
within short time period, with no damage to the existing
population using PTC techniques. Plant material can be
produced throughout the year without any seasonal limitation.
Large numbers of uniform and disease-free, virus free plants
can be produced from very small portions of the mother plant
due to the aseptic nature of tissue culture technique. The sterile
nature of in vitro cultures facilitates the exchange of
germplasm or plant materials even at international level
(Sharma & Sharma, 2013).Genetic resources of recalcitrant
seeds which are difficult to germinate, vegetatively propagated
plants, rare, threatened plant species, elite crop varieties and
some genetically modified plant materials can be efficiently
multiply and store in long term basis by using in vitro
techniques (Lidder & Sonnino, 2012).
5.3 Micropropagation and Cloning
In vitro clonal propagation method is commonly known as
micropropagation which helps to produce mass production of
plant propagules from any plant part or cell. Micropropagated
propagules are used to raise and multiply the stock plant
material in micro environment. Micropropagtion and cloning
of plant tissue based on different explants is commonly used to
conserve different endangered plants. In vitro propagation of
plants possesses huge potential in production of high quality
based medicines at the same time conservation of medicinal
plants. The in vitro plant regeneration/ micropropagation
program involved in several steps starting from initial shoot
development either directly from nodal part of explants or
357 Pathak and Abido

through indirect way of callus mediated de-differentiation of
shoot initials. Next, the shoot initials go for elongation and
developmental stage where small plantlets with well developed
shoots and root system will be generated for transplant in soil
(Castellanos et al., 2008; Sadeq et al., 2014a).
Micropropagation technique assists in the rapid, season
independent, continuous propagation, maintenance and storage
of rare and endangered plants by using any plant parts as
explant source (Sarasan et al., 2006; Chandra et al., 2010).
Several medicinally important plants are micropropaged using
several explants are listed in Table 1.
5.4 Somatic Embryogenesis and Organogenesis
The development of somatic embryo by the differentiation of a
single somatic cell or tissue to regenerate large number of
plants at the same time is very commonly used technique of
somatic embryogenesis in plant tissue culture. Somatic
embryogenesis and organ development through organogenesis
from various culture of explants are the most commonly used
technique applied to regenerate several endangered plants for
the purpose of conservation (Sadeq et al., 2014 a). Culture of
explant in suitable culture media helps to regenerate whole
plants either by following direct or indirect way of somatic
embryogenesis. In case of direct way of somatic
embryogenesis, plants directly develop from explants without
any intervening step of callus induction (mass of unorganized
cells) and dedifferentiation of callus towards organized growth
of plants as found in indirect way of somatic embryogenesis
Induction of somatic embryos from the cultured explants in
specialized culture media and their germination into whole
plantlet following several steps where different plant growth
regulators play important role (Sadeq et al. , 2014b). It has
great application in the rapid multiplication of important
medicinal plants those has considered as endangered. Table 2
presents a list of endangered plants regenerated through
somatic embryogenesis. Generally, the cultured cells or tissue
or regenerated shoots can be maintained by serial subcultures
at 4-8 week intervals for unlimited time period and plants can
be regenerated at any time to transfer in the suitable field
condition for conserving plant genetic resources. The culture of
germplasm by unlimited cultures has some disadvantages such
as risk of sudden loss of material due to human error or
infection, or genetic instability; this problem can be rectified
by restricting growth rate under culture condition.

Table 1 List of several micropropagated medicianally important plants.

Scientific name Importance Use Plant part Multiplication Ref

Aloe vera Anti-inflammatory Cosmetics, Leaf Micropropagation Hashem & Kaviani,
health benefit 2010
Asparagus Diarrhoea, asthma Therapeutic All parts Micropropagation Mehta &
adscendens and fatigue. Subramanian, 2005
Centella asiatica Terpinoids Antioxident, Leaf Micropropagation of Thangapandian et al.,
antidepressant whole plant 2012
medicines
Cunila galioides Volatile oils, Therapeutic Leaf and Micropropagation of Fracaro &
flower plant Echeverrigaray, 2001
Liriope Steroids Herbal Shoot Plant regeneration and Park et al., 2011
platyphylla meristem micropropagation
Melissa Essential oils Therapeutic Whole plant Micropropagation of Ghiorghita et al.,
officinalis terpinoids, part plant 2005
polyphenol
compounds
Olea europaea Phenolic Antioxidants, Fruit In vitro cultivation and Rkhis et al., 2006
compounds Health benefit plant regeneration
Cosmetic
Pelargonium Terpenoids Biopesticide Leaf Micropropagation Zuraida et al., 2013
radula antimicrobial
Phylanthus Alkaloids Herbal and Shoot part Shoot multiplication Xavier et al., 2012
amarus traditional
medicines
Psoralea Anti-inflammatory Therapeutic such as From Seeds Micropropagation Baskaran &
corylifolia Skin diseases Leucoderma, to all parts Jayabalan, 2008
leprosy, psoriasis
etc.
Pteris vittata Phosphate Arsenic Whole plant Micropropagation Shukla & Khare,
transporter detoxification 2012

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 The role of biotechnology in the conservation of biodiversity 358

Table 2 List of endangered and threatened plants regenerated via somatic embryogenesis and organogenesis by the process of plant
tissue culture.

Plant species Plant type Importance Explant used Multiplication References

Artemisia vulgaris Restricted Medicinal Leaf Organogenesis Borzabad et al.,
2010
Baliospermum montanum Threatened Medicinal Nodal bud Shoot differentiation Sasikumar et al.,
2009
Calligonum comosum Endangered Medicinal Node Shoot organogenesis Sadeq et al.,
and somatic 2014 b
organogenesis
Eleutherococcus senticosus Endangered Medicinal hypocotyl Somatic Choi et al., 1999
explants embryogeneis,plant
regeneration
Hedychium coronarium Endangered Medicinal Rhizome Somatic Verma &
embryogenesis Bansal, 2012
Heliotropium kotschyi Endangered Medicinal Node Shoot organogenesis Sadeq et al.,
2014 a
Lilium ledebourii Endangered Medicinal Bulb scale Somatic Bakhshaie et al.,
embryogenesis and 2010
plant regeneration
Psoralea corylifolia Endangered Medicinal hypocotyl Somatic Sahrawat &
segments embryogenesis Chand, 2001
Rauvolfia serpentina Endangered Medicinal Leaf Somatic Singh et al.,
embryogenesis and 2009
plant regeneration
Turbinicarpus Endangered Medicinal Medullar tissue Somatic Munoz & Garay,
pseudomacrochele discs embryogenesis and 1996
plant regeneration
Woodfordia fruticosa Rare Medicinal Shoot cuttings Organogenesis Krishnan &
Seeni, 1994

5.5 Growth Restriction
The practical of a plant tissue culture method in germplasm
conservation is to reduce the frequency of subculture during
the process of culture. Development and use of several slow
growth culture situations in modified culture media help to
reduce the subculture frequency (Bunn et al., 2007). The
addition and alteration of growth regulators, modification of
salt concentration of culture media, high or low concentration
of sugars in the growth media, addition of osmotically active
compounds and sometime modulation of some external factor
such as low temperature are used in slow growth strategy
(Reed &Chang, 1997). Tropical species are mostly cold
sensitive and have to store at high temperature. In vitro
cultivation of Musa sps. can be stored up to 15 months at 15ºC
(Banerjee & de Langhe, 1985). While other tropical species
such as cassava are more cold sensitive, so their shoots can be
stored at temperatures higher than 20ºC (Roca et al.,
1984).Various parameters influence the efficiency of in vitro
slow growth including type of explants, their physiological
state of culture entering, type of culture vessel, volume and
type of closures. In vitro slow growth storage technique is
routinely used for medium-term conservation of numerous
species both from tropical and temperate origin and
endangered species. However, in vitro conservation process for
medium and long term storage still need customization of
genetic stability with time and one single protocol for
conserving genetic diversity of the cultured materials are not
applicable. Sometimes genetic erosion during the program of
storage condition may hamper the total process, so special
guidelines for in vitro germplasm collection and storage of
genetic material in gene bank and botanical garden
establishment and management is very important (Pence et al.,
2002).
5.6 Cryopreservation
Cryopreservation is one of the biotechnological method of ex
situ plant conservation and applicable for long term storage of
plant genetic material. Cryopreservation is extremely helpful
method to conserve rare, endangered, threatened plant species
(Dussert et al., 1997; Zhao et al., 2008; Paunescu, 2009).
Moreover, for long-term storage of cultured plant materials
require the use of ultra-cold storage methods. This section
highlights the requirements for laboratory facilities and the
basic techniques. Moreover, most of the experimental systems
used in cryopreservation (cell suspensions, calli, shoot tips,
embryos etc.) contain high amount of cellular water and are
thus very sensitive to freezing injury. So, artificial dehydration
procedure is important step although classical techniques

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involve freeze induced dehydration, while new techniques are
based on vitrification (Zhao et al., 2008). The principle is
bringing the plant cell and tissue cultures to a non-dividing or
zero metabolism stage by subjecting them to supraoptimal
temperature in the presence or absence of cryoprotectants.
Classical freezing includes the successive steps such as
pregrowth of samples, cryoprotection, slow cooling (0.5-2ºC /
min ) to a determined prefreezing temperature (usually around
-40ºC, rapid immersion of samples in liquid nitrogen (LN),
storage, rapid thawing and recovery. It needs technical skill as
it has several steps such as freezing, storage, thawing, re-
culture of living plant cells during cryopreservation against
cryogenic injuries. Maintenance of cryogenic cultures in LN at
-196°C or in the vapor phase of LN at -135°C is in such a way
that the viability of stored tissues is retained following re-
warming. Various cryopreservation methods are being used
for various plant species such as vitrification, encapsulation–
dehydration and encapsulation vitrification (Stacey & Day,
2007). The genetic stability can be maintained during
cryopreservationthat has been proved by molecular marker
study (Liu et al., 2008). The cryopreserved tissue has
considered as safer, clean, disease free genetic stock for
international exchange (Feng et al., 2011).
5.7 In Vitro Gene Bank
The maximum possible genetic diversity of the particular
genetic stock can be maintained using in vitro gene bank
technique. Several International Organizations are engaged
potentially to conserve disease free, clean and elite class of
genetic stock and using this process mainly following slow
growth, in vitro techniques, cryopreservation of several stocks
together with routine analysis of genetic diversity. The
International Plant Genetic Resources Institute (IPGRI) as well
as the Consultative Group on International Agricultural
Research (CGIAR) Centers like the International Center for
Agricultural Research in the Dry Areas (ICARDA) is heavily
involved in the conservation of rare and endangered plant
species by maintaining in vitro gene bank (Reed et al., 2004;
ICARDA, 2014).
Several DNA marker based techniques are generally useful
methods in monitoring variability in rare and endangered plant
species. Another important fact is the simple and uniformly
available facility in the germplasm conservation program is
very important factor in monitoring gene bank at the
organizational level. Regular visual examination of tissue
culture derived plants growing in field or green house
condition must be conducted to identify any morphological
changes or to identify any somaclonal variations among them.
Selection and preservation of important desirable somaclonal
variations can be an important source of genetic variability
among the growing population.
In vitro derived genetic variation in somaclones are of
important source of genetic variability if it persists generation
after generation as genetic variant. A large number of
biotechnological approaches can be of useful technique in
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Pathak and Abido
determining the genetic variability among the germplasms. The
variability can be determined either using biomolecular
markers or by using DNA based markers such as restriction
fragment length polymorphism (RFLP), random amplified
polymorphic DNA (RAPD), amplified fragment length
polymorphism (AFLP), sequence characterized amplified
regions (SCAR), simple sequence repeat (SSR), inter simple
sequence repeat( ISSR), those are either PCR based or non-
PCR based techniques (Glaszmann et al., 1987; Khan et al.,
2012). Determination of storage conditions, provision of
inventory, evaluation of viability and verification of genetic
stability are very important components of gene bank structure.
DNA banking can be considered as a means of complimentary
method for the conservation of plant species together with
conventional ex situ approaches in preserving biodiversity.
6 Conclusions
Biodiversity is the very basis of human survival and economic
development. Ever increasing loss of biodiversity has posed a
serious threat to the survival of mankind. Worldwide one third
of the plant species are threatened due to several reasons. As
the conservation of biodiversity is a global concern, several
strategies has adopted in understanding and conserving plant
diversity throughout the world. Both ex situ and in situ
methods of biodiversity conservation are equally important.
The review presents the use of biotechniques in improving ex
situ conservation process to maintain biodiversity. It is now
well recognized that an appropriate conservation strategy for a
particular genotype requires combining approach of ex situ and
in situ techniques according to the need of the program. As it is
known fact that genetic variability is the main perquisite of the
survival of any plant species in their natural habitat, so study
of genetic diversity in conserved germplasm is important and
application of different biotechnological process playing a
promising role. In vitro plant propagation is a helpful
technique in the conservation of genetic diversity of all types
of plants (including rare, threatened and endangered plants) in
a rapid and reliable way by maintaining the same clone or
stock of plant material. It is just an efficient alternative method
of plant propagation technique. It goes well to the natural
process of plant conservation to restore the declined ecosystem
with tremendous applicability.
Acknowledgements
We would like to thanks colleagues at the College of Graduate
Studies, Desert and Arid Zone Sciences Program, Arabian Gulf
University, Manama, Kingdom of Bahrain for support and
encouragements.
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