African Journal of Biotechnology
African Journal of Biotechnology Vol. 2 (12), pp. 704-709, December 2003
ISSN 1684-5315 © 2003 Academic Journals
genetic conservation and sustainable use of bioresources
E. A.1, Nkang A. E.2* and Eneobong E.E.3
of Genetics and Biotechnology, University of Calabar, Nigeria.
of Botany, University of Calabar, Calabar, Nigeria.
of Applied Sciences, Cross River State University of Technology, Calabar,
author. E-mail: firstname.lastname@example.org.
17 November 2003
loss of Africa’s forests and bioresources is occurring at an alarming
rate, a consequence of increasing population pressure, agricultural land
degradation, urbanization and neglect. There is a growing recognition
worldwide that conservation and sustainable management of bioresources are
pressing priorities in the world today. The choice of conservation methods
and techniques depend on the objectives of the particular conservation
effort, the breeding system and behaviour of the species in question as
well as the available resources including funds, trained personnel,
infrastructure and technologies. The use of biotechnological tools and
“bioprospecting” will open new vistas in medicine, agriculture,
silviculture, horticulture, environment and other important issues. This
paper reviews some biotechnological tools that could be harnessed in
promoting conservation and sustainable use of bioresources.
Key words: Bioresources, genetic conservation, biotechnology.
refers to the total biological variation manifested as individual
plants, animals or their genes, which could be taken by man for use
as drugs, food, livestock feed, construction materials for shelter,
environmental protection, etc or in the development of improved
crops and animals for higher yield and tolerance to biotic and
abiotic stresses (Eneobong, 1997).
Man depends on these bioresources for his continued existence
and, therefore, he must use and preserve them for future generations
yet unborn. The concept of sustainable development indicates that
economic and environmental protection are inextricably linked and
that the quality of present and future life rests in meeting basic
human needs without destroying the environment on which life
rapid diminishing rate of Africa’s forests and bioresources have
been variously attributed to civil war, conversion of land for
agriculture, wild fires, poor management of available land,
uncontrolled search for food, fuel wood, medicine, construction
timber, overgrazing by cattle, displacement and loss of landraces,
lower yielding varieties, pests and diseases, pollution (e.g. acid
rain) and incomplete knowledge of the biology of many plants
especially the propagation genetics aspect and adaptability of many
forest plants (Eneobong, 1997).
and forest degradation are large-scale problems in developing
countries. In Nigeria,
the situation is made particularly pathetic with the frequent
increases in the prices of petroleum products, which have made a lot
of people to resort to the use of the cheaper and more steady
fuelwood. Between 1990
and 1995, the World Wildlife Fund (WWF) estimated that Africa lost
3.7 million hectares of forests every year, a deforestation rate of
0.7%, which is more than twice the world average of 0.3% and about
half of the world’s original forest cover has now disappeared.
The FAO (2001) also reported that global forest cover was
shrinking at a rate of around 9 million hectares per year.
These views are supported further by recent reports from the
World Resources Institute that while deforestation may have
increased in tropical Africa, it remained constant in Central
America and declined only slightly in tropical Asia
and South America.
we cannot do without exploiting the available bioresources to our
advantage, there has to be a balance between uses of resources and
their conservation. In this way, we would preserve an ecosystem,
which although altered would still be rich in bioresources and at
the same time would provide food and other needs as well as perform
vital environmental functions on a long term basis.
is necessary to conserve these naturally endowed resources which
apart from their direct usage by man, also serve several
ecological functions such as the control of flood, soil erosion,
landslides and hurricanes, maintenance of water quality, climate
amelioration and checking desertification (Okoro, 1994). A 1982 El
Salvador landslide that killed more than 1000 people was attributed
to deforestation. The hurricane Mitch that occurred in Central
America in 1998 was said to be compounded by about 30%
deforestation. The endangered species and threatened crops deserve
urgent and special attention. For example, the world’s only
remaining wild Orang-utangs would be gone for good if Indonesia’s
remaining natural forest is destroyed. The Afi River Forest Reserve
(Cross River State, Nigeria) is located in one of Africa’s
diversity ‘hotspots’. This area is also one of West Africa’s
three ‘deforestation hotspots’, recognized by the Tropical
Ecosystem Environment Observations by Satellite program (TREES,
European Commission). The Afi reserve is home to the newly
subspecies of chimpanzee and the Cross River Gorilla, Gorilla gorilla diehli, recognized as a distinct and critically
endangered subspecies by the Primate Specialist group of the
International Union for the Conservation of Nature (IUCN) Species
Survival Commission in February 2000. Several species of birds and
monkeys in Africa would have become extinct by now if not for the
intervention of wildlife conservationists, as there is now greater
international willingness to ‘pay’ for the conservation of
Biotechnology can be defined as any technique that uses living organisms or parts thereof to make or modify a product, improve plants or animals or develop microorganisms for specific uses (Alhassan, 2001). It is invaluable in research on conservation of bioresources. Although modern biotechnology is a newly introduced science (less than 50 years old) its impact has greatly excited the imagination and provoked the concern of almost every part of the society worldwide (Eneobong, 2003). Developments such as the tomato that can be frozen and the cassava and other agricultural crops that have been genetically engineered for insect and virus resistance and which are in or near commercial release are simply spectacular by any standard. The richness of plant and animal diversity in developing countries is a major asset in agricultural development and, therefore, the conservation of such resources is fundamental to the progress and usefulness of biotechnology.
Conservation of genetic resources
conservation of plant genetic resources can best be achieved through an
appropriate combination of in situ
(in natural or original areas) and ex
situ (in artificial habitat or habitat different from the original
one) methods (IPGRI, 2001). The choice of conservation methods and
techniques will depend on the objectives of the particular conservation
effort, the breeding system and seed behaviour of the species in
question as well as on the available resources including funds,
infrastructure and technologies (Perrino, 1990; Eneobong, 1997).
Generally plants with orthodox seeds (high tolerance of low temperature
storage conditions) are best preserved ex
situ, under medium or long term conditions as comparatively dry
seeds stored at low temperatures (Ng, 1991).
Plants that produce
recalcitrant seeds (intolerant of desiccation and low temperatures)
could be preserved as ex situ live-gene
banks (or gene libraries) or by in
vitro conservation methods of enforced reduced growth storage. Plant
resources are also routinely preserved in
situ in parks, reserve areas and rangelands.
section of the paper will discuss in
vitro methods for storage as well as the cryopreservation of
embryos, seeds, protoplasts and other materials in long-term liquid
nitrogen base-storage systems. Many plants, especially forest plants,
are extremely difficult to propagate through conventional means since
they are frequently polyploids and aneuploids or produce seeds with
little or defective endosperms. In Nigeria, very little is known about
the biology of some of these “orphan” plants. There is therefore the
risk of losing some of these plants due to industrialization and
urbanization, characterized by rapid deforestation, uncontrolled
logging, burning and uncontrolled search for food and other non-timber
forest products. Many of these plants constitute an important component
of the diet in many West African countries (e.g. Gnetum
africanum, Treculia africana (breadfruit), Irvingia
gabonensis) and costly timbers (e.g. Diospyros
mespiliformis (iroko), Entandrophragma
cylindrica (mahogany) and Chlorophora
excelsa). Plant tissue culture provides a method for the mass clonal propagation of such
materials, as well as serving as a tool for their germplasm
This refers to in vitro mass production of plant propagules from any plant part or
cell. Such propagules are used to raise whole plants. The principal
Axillary budding: The induction of adventitious buds on non-meristematic
tissue (that is, inducing a shoot where one should normally not exist).
Somatic embryogenesis: Where individual cultured cells or small
groups of cells undergo development resembling that of the zygotic
embryo. The embryoids produced can be used to produce whole plants.
attraction of micropropagation, as an alternative to other propagation
methods, lies in its ability to multiply elite clonal material very
rapidly. More than 1000 plant species have been micropropagated,
including more than 100 forest tree species
(FAO, 2001). Work done with some crop species indicates the
possibility of encapsulating somatic embryos to form artificial seeds,
which can then be handled like conventional seeds. Such propagules may
be used in forest plantation establishments.
exploits the “totipotency” nature of plant cells and tissues. The
explants are made to form callus under appropriate nutrient
environments. Numerous clonal plants can be obtained from sub-cultured
callus or from embryoids, which are then hardened and transferred into
potted soil in nurseries. In this way planting propagules can be
provided for rare or threatened plant species as well as for plants with
inviable or difficult-to-germinate seeds. Furthermore, tissue culture
techniques can serve as an enhancing tool in plant breeding for the
rescue of defective hybrid embryos, caused by post-zygotic
incompatibility during crossing (Enebong and Okonkwo, 1994). Nutrients
provided in the culture medium will perform the function of the
malformed endosperm. Embryo cultures also adopt the nutritional and
physical requirements for embyonic development to bypass seed dormancy
(thus shortening the breeding cycle) and seed sterility and to provide
micro-cloning material (Hu and Wang, 1986).
conservation of vegetatively propagated crops (e.g. banana, plantain,
yam, cassava etc) forest species especially those with recalcitrant
(hard to store) seeds (e.g. mango, cocoa, Symphonia
gabonensis) in live gene- banks in fields
poses tremendous problems in terms of required land space and labour
input during annual or perennial replanting, testing and documentation.
Such collections are also exposed to threats by biotic and abiotic
stress agents. Consequently, in vitro conservation is recommended, at least as a supplement to
field collections, as long as an adequate protocol for micropropagation
has been worked out for the species.
advantage of in vitro or
reduced growth storage include little space necessary in growth rooms
for maintaining thousands of genotypes and the absence of diseases and
pest attack in culture vessels. Furthermore, in
vitro storage eliminates the need for long and frustrating
quarantine procedures during movement and exchange of germplasm.
any part of the plant could be used as explant in establishing cultures
for storage, although the best results have been obtained using apical
meristems, axillary buds, embryos and gametes. Normal in
vitro cultures use media like Murashige and Skoog and Arnold and
subcultures into fresh media are necessary. Excised plant tissues,
organs, or cells are usually cultured on these media. The culture medium
could be liquid or solidified using agar. Under these conditions the
cultures rarely last longer than a few months, requiring transfers into
fresh media to maintain optimal growth.
in vitro storage the growth of the culture is slowed down through
one or a combination of several methods namely
Reducing the concentrations of the minerals or by using media
with lower salt concentrations (Ng and Ng, 1991).
Using low incubation temperatures (Dale, 1980; Henshaw et al., 1985; Ruredzo and Hanson, 1991).
By the addition of osmotica (Henshaw et
al., 1980, Ng and Ng, 1991; Ruredzo and Hanson, 1991).
Reduction of the gas pressure in culture vessels
and Staby, 1981).
By varying the light regime (Mullin and Schelegel,
plants are normally stored either in active gene banks containing
material that is kept ready for distribution, evaluation or exchange; or
as base collections containing duplicates that are kept for future use
(long term) or “emergency” material in case of loss from the active
gene banks. Base collections can be maintained under very cold conditions
in high tech ultra low temperature freezers. Cryopreservation is an
attractive alternative for the storage of base collections and involves
the freezing of plant material, usually to the temperature of liquid
nitrogen (-196oC), at which point cell division and
consequently growth and all other biological activities are completely
arrested. This must be done
in a manner that viability of the stored material is retained and
biological functions and growth can be reactivated after thawing (Towill,
1991; De Smet, 1995). Liquid
nitrogen storage is useful for the preservation of various types of
plant material including whole seeds, embryos, suspension cells, callus,
protoplast cultures, gametes and meristems.
techniques of freeze-preservation in liquid nitrogen have been modified
in several ways to minimize freeze damage, there are three major steps
The material to be frozen needs to be cryoprotected from freeze-damage
by treatment with cryoprotectants.
The commonly used chemicals for this purpose include dimethyl
sulphoxide (DMSO), ethylene glycol, glycerol and proline, used at
concentrations usually less than 10% (w/v), to enhance the survival of
hydrated tissues or cells during freezing. These chemicals cause changes
in cell permeability, freezing point and responses to stresses of
freezing and thawing. Cryoprotectants can be added before freezing or even at lower
temperatures during the freezing process.
For the former, incubation for a few hours may be necessary
before initiating the freezing process. Some pretreatment methods, which
have been found to be successful in some species when used with
cold treatment for callus or cell suspension cultures
preculture of shoot tips with 0.25-0.75 M sucrose and 5% DMSO (Dereuddre
et al., 1987), and
cooling under osmotic stress induced with mannitol
or sorbitol (Towill,
A two-step process of cooling is recommended for most plant material,
since continuous slow cooling below –33oC to –40oC
leads to considerable loss of viability in many plants (Towill, 1991).
In practice, the initial cooling down to –40oC may
be fast (several hundred oC/min) or slow (0.5-2oC/min)
depending on plant genotype, before the storage in liquid nitrogen (Ng,
1991). Freezing may be
initiated by the induction of a nucleation with a small ice crystal
achieved by briefly touching the outside of the cryotube with LN-cooled
forceps (Towill, 1991) or, as in bananas and plantains by plunging the
cryotube for a few seconds in liquid nitrogen at temperatures near zero
and then slow cooling to -40oC (De Smet, 1995).
Rapid warming is necessary during the thawing process and the tube
containing the cryopreserved material can be dipped directly into warm
water (30-40oC) for a few seconds to melt the ice.
In the cases of cultured cells, callus and meristems, the samples
are then diluted with liquid culture medium to remove the
cryoprotectants and the viability of the cryopreserved material can be
tested through regeneration or germination in the case of seeds.
Animal genetic resources
genetic resources, like plant genetic resources need to be conserved for
future generations. The use of artificial reproduction is a very useful
tool in conservation of endangered species. It should, however, be made
to complement the conventional methods of breeding.
Some of the biotechnological methods used for production and
conservation of animal genetic resources are summarized below:
procedure is similar to what has been described already for plants.
Materials such as cells, tissues, gametes, oocytes, DNA samples etc are
stored in a genetic databank for future use.
used in the production of embryos in
vitro include splitting and cloning of embryos, marker-assisted
selection, sexing of embryos and transfer of new genes into an embryo
(First, 1992). Cloning in
animals is enhanced by nuclear transplantation, a method used to produce
a large number of viable identical embryos and offspring of desirable
genotype in cattle, sheep, rabbits and swine.
The procedure involves the separation and transfer of nuclei of a
valuable embryo at a multicellular stage into enucleated oocytes at
metaphase II followed by serial cloning (First, 1992).
Embryo Culture and Transfer
technique is used to introduce fertilized embryos into surrogate
mothers. Sometimes closely
related species can be used to produce the offspring of an endangered
species. The great majority
of commercial embryo transfer is done with cattle for strictly economic
reasons since the economic value of production per head is much higher
for cattle (and buffaloes) than for other farm animal species (Serdel
and Serdel, 1992).
technique is useful in livestock farming.
Cryopreserved sperm from selected males are thawed and introduced
into ovulating females.
Intracytoplasmic sperm injection
sperm from selected males are microinjected
directly into the oocyte.
These biotechnological methods offer many advantages to conventional captive breeding procedures. Firstly, less stress is experienced since the animals do not have to be moved around. Secondly, the problem of space for keeping the animals is also solved since samples can be taken in the wild. Thirdly storage of genetic resources will help to preserve biodiversity and counter the effect of genetic drift on small populations. Fourthly, even if an animal dies, its genes will still be available for future breeding work. Also gametes can be extracted from animals that have been dead for up to 24 h and cryopreserved for future use. The main disadvantage is that sometimes preserving only the DNA samples may not be enough to conserve the entire animals as many animals need to learn behaviour (which may not be in the genes) in order to survive. Moreover, the use of biotechnological tools for endangered species is still at a very early stage and is very expensive.
use of bioresources
utilization or exploitation of bioresources has led to genetic erosion,
desertification and a general threat to the survival of man.
Sustainable use of bioresources demands that while utilizing the
resources so generously placed by nature at our disposal, we should try
not to be cruel to the environment and our children yet unborn.
Biotechnology provides methods through which a balance between the
economic exploitation of bioresources and their conservation for the
future can be achieved. Some
of the techniques of biotechnology and their applications for the
sustainable use of bioresources are summarized below:
Plant Cell Tissue Culture
refers to the culture of explants usually embryos, seeds, cells (virtually
any part of the plant) on specific media composed of major and minor
mineral salts, iron, vitamin and a carbohydrate source (usually sucrose)
and subsequently regeneration of whole plants therefrom. It has found
(i) Mass clonal propagation; disease elimination
(mainly viral); germplasm exchange; in
vitro conservation and cryopreservation of seeds, embryos, suspension
cells, meristerms and other suitable plant parts. It is especially useful
for threatened plants, and crops with recalcitrant seeds and seedless
Embryo culture for overcoming postzygotic incompatibilities.
Anther/pollen and ovary cultures for fast production of homozygous
plants through embryogenesis and chromosome doubling.
Haploids could be useful for isolation of desirable recessives.
production of plant secondary metabolites.
Generation of variability in somaclones
Protoplast isolation, fusion and culture. This is useful in
overcoming prezygotic incompatibilities in crossing.
Biological Nitrogen fixation. Used for development of
biofertilizers; improvement of the capability of free-living N-fixing
bacteria and development of farming systems using green algae and Azolla.
Use of Molecular Markers:
and animal breeders use markers to aid selection for desirable/beneficial
genotypes. These molecular
markers are based on DNA variation and can be grouped into two:
Those based on restriction and hybridization techniques and include
restriction fragment length polymorphism (RFLP), which is costly,
cumbersome and use isotopes in blotting and is thus avoided by many
laboratories (Eneobong 2003).
Those based on the polymerase chain reaction (PCR) is used for gene
amplification and include:
Random amplified polymorphic DNA (RAPD), otherwise called Arbitrary Primed
PCR (AP- PCR). Here, a pair
of DNA primers are designed to hybridize to opposite strands of the
genomic DNA, acting as primers for the in
vitro synthesis of the intercalated DNA sequence (Mignouna et
al., 1999; Eneobong, 2003).
DNA Amplification finger printing (DAF).
It uses DNA primers to generate amplified products through the PCR.
Such products can be stained in mercury during gel electrophoresis. The
method is useful in germplasm and phylogenetic studies (Eneobong, 2003).
Amplified fragment Length Polymorphism (AFLP). This is a genetic
fingerprinting technique based on detection of selected genome restriction
fragment by PCR amplification. The
method is useful for detection of genetic variation in
vitro (Ubi et al.,
Gene Transfers/Genetic Transformation:
modern techniques for gene transfer are based on the natural process of
transformation. They are mainly recombinant DNA technology plus tissue
culture, aided by several molecular biology tools such as gene isolation,
cloning and vector construction. The technique is used for production of
transgenic organisms. Examples
Agrobacterium-mediated transfer which is quite successful for
dicots but not monocots (Eneobong, 2003).
Direct DNA uptake. This has found application more in animals than
in plants. The first attempt to transfer foreign DNA in animals was done
in mice by microinjection. The first transgenic sheep and pigs were
reported in 1985 when a mouse metallothionin growth hormone (mMThGH)
fusion gene was transferred into sheep.
Since then, many transgenic farm animals have been produced on a
routine basis. Examples are
transgenic fish that grows 2-3 times faster than normal and is
cold-tolerant was produced by microinjection of desired DNA into oocytes,
transgenic cattle, sheep, swine and rabbits produced by microinjection of
desirable gene into zygote to produce faster-growing animals with better
meat quality, transgenic goats and sheep which produce human milk because
of the transfer of human genes into such animals, and transgenic chicken
which grow faster and are tolerant to viral diseases because of the
transfer of growth hormone gene as well as a gene that increases viral
resistance based on interference (Bazer, 1992; Fox, 1992; Forano and Flind,
Particle mediated gene transfer, using gene gun.
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