APPLICATION OF BIOTECHNOLOGY IN PGR

CONSERVATION AND MANAGEMENT

Crop germplasm collections are assemblies of genotypes or populations
representative of cultivars, genetic stocks, wild species etc., which are
maintained in the form of plants, seeds, tissue cultures etc . Functionally, plant
genetic resources (PGR) can include landraces, advanced/improved cultivars
and wild and weedy relatives of crop plants (either domesticated, semi-
domesticated or non-domesticated).
The activities that relate to conservation use of PGR include: exploration and
collecting; characterization and evaluation; conservation, assessment of
variation and identification of useful genes; exchange and genetic
enhancement. Exploration is a preliminary survey of an area where germplasm
collecting is to be carried out, however due to resource constraints, most often
exploration and collecting are carried out simultaneously
Systematic characterization and evaluation of material. Conservation includes
the management, preservation of known genetic resources. Conservation of
PGR can be divided into two approaches, ex situ and in situ. Ex situ
conservation maintains germplasm outside its original habitats in facilities that
have been specifically created such as the seed, field and in vitro genebanks or
botanical gardens
The International Board for Plant genetic Resources (IBPGR) was established in
1974 to promote and coordinate the work on plant genetic resources globally
and was linked to the Food and Agricultural Organization of the United Nations
(FAO) for administrative purposes. During the last 20 years, along with many
achievements to its credit, IBPGR has now grown into a fully independent
institute, the International Plant Genetic Resources Institute (IPGRI) and is one
of the 21 institutes in the Consultative Group on International Agricultural
Research (CGIAR) system.

IPGRI's mandate is to advance the conservation and use of PGR for the benefit
of present and future generations. IPGRI does not have either genebanks or
laboratories. It works by collaborating with different partners who have an
interest in conserving and using PGR
IPGRI recognizes that biotechnology provides some important tools for
effective conservation and use of PGR. These tools include tissue, cell or organ
culture, molecular genetic techniques, serology, embryo rescue techniques
and DNA extraction and sequencing
HC SHARMA

THE ROLE OF BIOTECHNOLOGY

So far we have seen that the subject area for germplasm comprises of
exploration and collecting, conservation, characterization and evaluation,
exchange of germplasm, and genetic enhancement. In functional terms, these
mainly constitute in vitro technology and molecular techniques. The former,
involving tissue culture, preservation, and regeneration of plant material are
useful in collecting, conserving, germplasm health and exchange etc. We have
also briefly seen what is IPGRI and about its role in the conservation and use of
PGR globally. Now we will attempt to review some of the available
biotechnologies and the ways in which these can assist in carrying PGR related
activities effectively and efficiently.

Exploration and Collecting

Germplasm collecting generally involves collecting samples of seed from
populations of species. However, collecting clonally propagated plants and
species with recalcitrant seeds is often problematic. This is because often the
material collected is bulky, can deteriorate fast and can be infested with pests.
Due the advances in tissue culture a range of techniques are now available for
in vitro collecting of germplasm of such problem species

Characterization and Evaluation

So far we have seen the need and urgency for PGR collecting. Historically
germplasm collecting and conserving have had significance in elucidating
taxonomic status and evolutionary relationships between and within species.
Though it is an important role for germplasm collections, the main justification
for genetic resource conservation is there for use in crop improvement
Four areas of germplasm "characterisation" in which biotechnology can be
used have been identified: a) identification of genotypes, including duplicate
accessions; b) "fingerprinting" of genotypes; c) analysing genetic diversity in
collections or in natural stands; and d) assembling a core collection (Dodds and
Watanabe 1990).
This rapid progress has resulted in the development of a number of methods
to detect variation in nucleotide sequences have been developed in recent
years and some of these are:
1) Restriction fragment length polymorphism (RFLP): Its resolution is at
genomic or chromosomal level and principally it is a marker technique, mainly
useful in gene mapping and marker assisted selection. Technically complex and
needs use of short-lived radio isotopes (although some research on the use of
non-radioactive labels is under way). So routine application of RFLPs in large
scale genetic diversity analyses may be difficult.
2) Random amplified polymorphic DNA (RAPD): This is a type of marker, using
polymerase chain reaction (PCR) technology, basically simple, quick, requires
only small amounts of DNA, involves no radioactivity and is suitable for large
samples. Resolution is at genomic level and it is a typical marker technology.
RAPDS segregate in Mendelian fashion and are useful in population genetic
studies, in locating and manipulating genes of interest, to identify somatic
hybrids at an early stage, to monitor the levels of genetic diversity between
and within populations, to 'fingerprint' individual accessions.
3) Variable number of tandem repeats (VNTRs): These are serial repeats of a
core DNA sequence, depending on the size it can be micro or mini satellites. In
a single locus situation, VNTRs are useful for identification of individuals,
paternity analysis. In multilocus situation, they are useful for marker assisted
selection, studying relatedness of individuals, and genetic variation.
4) Polymerase Chain Reaction (PCR/sequencing): Studies the base sequence
of known genes, gene is amplified, sequenced revealing the composition. PCR
has the highest level of resolution, as the differences are measured at base
level. Significant extent of automation of the technique is possible and this
assists large scale application. Useful in population studies, taxonomy and/or
phylogenetics. PCR is a powerful technique for identification of infraspecific
genetic variation (VNTR and PCR techniques offer distinct advantages over
RFLP, isozyme or morphological analysis (Swennen 1990).
5. Allele specific polymerase chain reaction (ASPCR)
This technique uses allele-specific oligonucleotide primers in PCR
amplifications. It is a fast, accurate, sensitive technique but main difficulty is
the development of allele-specific reagents. This been used for identification of
genotypes (Shattuck-Eidens, et al. 1992). 6) Denaturing/temperature gradient
electrophoresis (DGGE/TGGE): This technique detects differences down to
single base substitutions so it is useful in studying mutations. It is mainly used
in population studies.

Conservation of Germplasm
As alluded to in the beginning, there are two approaches to conservation of
PGR: ex situ and in situ. It is important to emphasize that these two
approaches are not mutually exclusive, but are indeed complementary. The
work of conserving a genepool should employ a combination of methods, from
nature reserves to genebanks, The appropriate strategy and the balance
depends on factors such as the biological characteristics of the genepool,
infrastructure and human resources, number of accessions in a given collection
and its geographic site

Ex situ Conservation of Seeds

It is well known that under cool and dry conditions orthodox seeds are viable
for long periods. Seed longevity being, to some extent directly proportional to
the storage temperature, humidity and seed moisture content. If seeds are
maintained under such conditions, the life processes in seeds are minimized so
that they could be stored for a number of with little loss in genetic diversity,
genetic integrity and viability

Ex situ Conservation of Vegetatively

Propagated Material
There are a number of important plant species, including important staple food
crops and fruits such as cassava, potato, sweet potato, taro, yam, apple,
banana, citrus, etc., which are difficult or impossible to propagate by seed and
cannot be conserved as seeds. Generally, these are conserved in field
genebanks.
Tissue culture:
Possibilities now exist conserve PGR as tissue cultures (Withers and Engels
1990). For some species, the in vitro conservation may be the only option
available. Though tissue culture offers great potential for conservation
germplasm of vegetatively propagated material and species with recalcitrant
seeds, two things have been of major technical hindrance to it. Firstly, the
genetic instability of the material conserved as tissue culture due to
somaclonal variation at the time of regeneration of the tissue into seedlings.
Secondly, the length of storage as tissue has been limited. Significant work is
being done on both the aspects and for some species, tissue culture
maintenance is relevant due improved techniques resulting in low levels of
somaclonal variation

Cryopreservation:
Theoretically, cryopreservation i.e. using liquid nitrogen (either by immersion
or in vapour phase) as storage medium, is ideal for long term storage since it
virtually suspends all the metabolic activities in any living tissue, be it seed, cell
suspensions, callus, cultured tissue, pollen or a shoot tip

Synthetic Seeds:
Another promising method for the conservation of species that are clonally
propagated or species with recalcitrant seed is the possibility of producing the
so called 'synthetic' or 'artificial' seeds and conserving them as true seeds
(Engels, 1993). This involves encapsulation of shoot-tips and somatic embryos
in semi-solid material which serves as artificial seed coat and endosperm and
producing 'beads'.

genebanks:
So far we have discussed different components of an entirely in vitro-based
conservation scheme for clonally propagated and other problem crops. There
is need to put al these components together - protocols for tissue culture,
successful regeneration, transfer to soil (the protocols which differ from
species to species, sometimes there are genotypic differences), genetic
stability, cryopreservation of cultured material either by vitrification or by
encapsulation. When this is achieved we have a viable long term conservation
strategy for PGR for an in vitro genebank.

In vitro genebanks:
So far we have discussed different components of an entirely in vitro-based
conservation scheme for clonally propagated and other problem crops. There
is need to put al these components together - protocols for tissue culture,
successful regeneration, transfer to soil (the protocols which differ from
species to species, sometimes there are genotypic differences), genetic
stability, cryopreservation of cultured material either by vitrification or by
encapsulation. When this is achieved we have a viable long term conservation
strategy for PGR for an in vitro genebank.

Safe Movement of Germplasm

It is important that all accessions in the genebank should be available to all
those who wish to use them, either in crop improvement or for other studies.
No country is self sufficient in PGR that is required for crop improvement
programmes of the country.
Biotechnology has played an important role in assisting safe distribution of
PGR through exchange of germplasm as disease-free cultures (Delgado and
Rojas 1993; Dodds and Watanabe 1990; Frison 1981; IBPGR 1988;Ng 1988).

Biosystematics and Evolution

Biosystematics at both interspecific and infraspecific levels are essential for
proper classification of the material conserved, to determine breeding
behaviours such incompatibility etc., for effective conservation and use of PGR.

Use of germplasm
One of the major objectives of conservation of PGR is to make genetic
diversity available for immediate or future use. Abundant evidence exists that
it is necessary to preserve a wide range of diversity in order to meet the
current crop improvement needs. However, it is also evident that the widest
possible range of genetic diversity has to be conserved in order to meet future,
as yet unknown, needs (Hodgkin and Debouck, 1992). PGR programmes are
expected to promote and facilitate the use of germplasm through:
maintenance of sufficient healthy and readily-accessible and adequately
characterised/evaluated material; and proper documentation of the relevant
information.

RFLPs have been used for fingerprinting, to generate genetic maps (West, et al.
1989) and to enable the identification of specific genotypes and agronomic
traits (Dodds and Watanabe 1990; Rao and Grandillo 1992). High density RFLP
maps provide an opportunity to resolve complex traits into their individual
genetic components. It might then be possible to treat these characters as
single gene traits.

Reference
ResearchGate
Article published by
Ramanatha V Rao & K W walley
GRSV Consultancy Service, Bengaluru