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KEYWORDS

Biodiversity

Biotechnology

Endangered plants

Ex situ

Genetic diversity

In situ

Plant Tissue Culture

In Vitro Gene Bank

ABSTRACT

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

their life support. It has been estimated that one third of the global plant species are threatened in

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,

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

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

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,

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

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.

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

THE ROLE OF BIOTECHNOLOGY IN THE CONSERVATION OF

BIODIVERSITY

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

Peer review under responsibility of Journal of Experimental Biology and

Agricultural Sciences.

* Corresponding author

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

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ISSN No. 2320 – 8694

Production and Hosting by Horizon Publisher (www.my-vision.webs.com/horizon.html).

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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

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

353 Pathak and Abido

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.

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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

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

The role of biotechnology in the conservation of biodiversity 354

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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

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.

355 Pathak and Abido

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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

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

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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,

health benefit

Leaf Micropropagation Hashem & Kaviani,

2010

Asparagus

adscendens

Diarrhoea, asthma

and fatigue.

Therapeutic All parts Micropropagation Mehta &

Subramanian, 2005

Centella asiatica Terpinoids Antioxident,

antidepressant

medicines

Leaf Micropropagation of

whole plant

Thangapandian et al.,

2012

Cunila galioides Volatile oils, Therapeutic Leaf and

flower

Micropropagation of

plant

Fracaro &

Echeverrigaray, 2001

Liriope

platyphylla

Steroids Herbal Shoot

meristem

Plant regeneration and

micropropagation

Park et al., 2011

Melissa

officinalis

Essential oils

terpinoids,

polyphenol

compounds

Therapeutic Whole plant

part

Micropropagation of

plant

Ghiorghita et al.,

2005

Olea europaea Phenolic

compounds

Antioxidants,

Health benefit

Cosmetic

Fruit In vitro cultivation and

plant regeneration

Rkhis et al., 2006

Pelargonium

radula

Terpenoids Biopesticide

antimicrobial

Leaf Micropropagation Zuraida et al., 2013

Phylanthus

amarus

Alkaloids Herbal and

traditional

medicines

Shoot part Shoot multiplication Xavier et al., 2012

Psoralea

corylifolia

Anti-inflammatory

Skin diseases

Therapeutic such as

Leucoderma,

leprosy, psoriasis

etc.

From Seeds

to all parts

Micropropagation Baskaran &

Jayabalan, 2008

Pteris vittata Phosphate

transporter

Arsenic

detoxification

Whole plant Micropropagation Shukla & Khare,

2012

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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

and somatic

organogenesis

Sadeq et al.,

2014 b

Eleutherococcus senticosus Endangered Medicinal hypocotyl

explants

Somatic

embryogeneis,plant

regeneration

Choi et al., 1999

Hedychium coronarium Endangered Medicinal Rhizome Somatic

embryogenesis

Verma &

Bansal, 2012

Heliotropium kotschyi Endangered Medicinal Node Shoot organogenesis Sadeq et al.,

2014 a

Lilium ledebourii Endangered Medicinal Bulb scale Somatic

embryogenesis and

plant regeneration

Bakhshaie et al.,

2010

Psoralea corylifolia Endangered Medicinal hypocotyl

segments

Somatic

embryogenesis

Sahrawat &

Chand, 2001

Rauvolfia serpentina

Endangered Medicinal Leaf Somatic

embryogenesis and

plant regeneration

Singh et al.,

2009

Turbinicarpus

pseudomacrochele

Endangered Medicinal Medullar tissue

discs

Somatic

embryogenesis and

plant regeneration

Munoz & Garay,

1996

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

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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