INDIRA GANDHI KRISHI VISHVAVIDHYALAYA

AN ASSIGNMENT

ON

Germplasm maintenance and storage

In situ and Ex situ conservation of germplasm

Course no. : GP-610

Submitted to:

Dr. A.K. Sarawgi

Dr. S.S. Rao

Dr. S.K. Nair

Deptt. of Genetics and Plant Breeding Submitted by:

Kamini Dhiwar

Ph. D. Scholar, 1st Year

Deptt. of Genetics and Plant Breeding

COLLEGE OF AGRICULTURE, RAIPUR

 GERMPLASM MAINTENANCE AND STORAGE

What is germplasm?
 The sum total of hereditary material i.e. all the alleles of various genes, present in a crop
species and its wild relatives
 Germplasm is a genetic substance that can be passed from one generation to another, sexually
or somatically
 Genotypes of particular species, collected from different sources and geographical origins, for
use in plant breeding to develop new cultivars.

Genetic
stock

Wild and
Landraces weedy
relatives

Germplasm
or plant
Parental genetic
resources Released
liness of
variety
hybrids

Primitive Farmers
cultivars varieties

Germplasm storage:-

 Germplasm storage is also known as germplasm conservation. Germplasm conservation is the

most successful method to conserve the genetic traits of endangered and commercially
valuable species. Or germplasm conservation is defined as the maintenance of gerplasm in
such a state that there is minimum risk for its loss ant that either it can be prepared for
planting with relative ease.
  The main objective of germplasm conservation is to preserve the genetic diversity of selected
plants or genetic stock for its utilization at any time in future.
 Germplasm conservation is a key factor in preserving knowledge about extinct, wild, or other
living species because genetic diversity leads to the extinction of older generation genetic
material. It is mostly concerned with ensuring the secure handling and proper preservation of
germplasm from commercially valuable plants by collecting each taxon's seed.

Method of Germplasm Storage:-

 There are several ways of conserving plant genetic resources of crop plants. The cheapest and
most convenient is to store the seeds. However, not all crops can be preserved in this way,
either because they are vegetatively propagated or do not produce seeds or because the
periods of the seeds viability may be extremely short under storage (Ng, 1988).
 There are, however, conservation methods that depend on factors such as the storage organs
or propagules, the extent and geographical distribution of genetic diversity and the
availability of suitable storage facilities. Germplasm storage or conservation is basically two
types-
 In situ conservation
 Ex situ conservation

In situ conservation Ex situ conservation

In situ conservation:-

 This is the preservation and protection of genetic resources in their natural habitat (Ford-
Lloyd and Jackson, 1986). It is the continuing maintenance of a plant population within the
ecological community in which it forms a part as well as in the environment to which it is
adapted. This means of conservation aims at achieving stability by maintaining self-
perpetuating population in natural system.
 In situ conservation enables many more species to be conserved under conditions that allow
them to continue to evolve. Conservation of plant in situ allows the preservation of cultivated
and wild species without the need for big expenditure of the area to be conserved.
 One great advantage of this conservation method is that species can continue to evolve,
allowing the appearance of new recombinant forms.

Specific goals of the in situ conservation:-

 Ensures continued access to populations for research and availability og germplasm.

 Ensure availability of material of target species or populations for use by local people.
 Conserve species which cannot be regenerated outside their natural habits.
 Enable some degree of conservation of associated species which may or may not be of known
economic value and are of importance for maintaining a healthy ecosystem.
 Minimize human threats to genetic diversity and support actions that promote such diversity
in target population.
 Minimize the risk of genetic erosion from demographic fluctuations, environmental variation
and catastrophes.

Types of in situ conservation-

1. Monitored at international level

 Global heritage centres

 heritage centres

2. Monitored at national level

 On- farm conservation
  Biosphere reserves
 Gene sanctuaries
 Sacred grooves

On-farm conservation

On-farm conservation is the conservation of crops and their wild relatives, livestock, and the
agro ecosystems in which they occur. Agro ecosystems include home gardens, crop fields,
agroforestry systems, fallow fields and grazing lands. Agricultural biodiversity, or agro biodiversity,
is that component of biodiversity that contributes to food and agricultural production. In situ
conservation on farm is important for maintenance of agro biodiversity.

Indigenous resource management systems and agricultural practices play an important role in
the maintenance and diversification of domesticated plants and animals (McNeely et al., 1995). Low-
input agricultural systems are important sources and custodians of agro biodiversity. Farmers and
pastoralists maintain a tremendous diversity of crop and livestock varieties around the world.
Indigenous knowledge, skills and practices of farmers play an important role in the conservation
and management of agricultural biodiversity.

They are better options for building the scientific basis of in situ conservation of agro
biodiversity on-farm (Deribe et al., 2002). For instance, the farmers’ indigenous knowledge and
practices in germplasm selection, storage and exchange are major elements in the conservation of
agricultural biodiversity through community gene banks. The expansion of large-scale/modern
agricultural systems, in which relatively a few improved varieties have replaced many farmers’
varieties, has caused erosion of agricultural biodiversity.

Therefore, there is a need for basing the rural development strategy on traditional farming
systems, knowledge and agro ecological techniques in order to ensure the maintenance and continual
use of the diverse genetic resources associated with traditional agricultural systems (Miller et al.,
1995). Farmer-based on-farm conservation of agro biodiversity has been found a more successful
approach.

Biosphere reserves:-
 The idea of biosphere reserves was initiated by UNESCO in 1973-74 under its Man and
Biosphere (MAB) programme. Biosphere Reserve (BR) is an international designation made by
UNESCO for representative parts of natural and cultural landscapes extending over large area of
terrestrial of coastal or marine ecosystem or a combination thereof. BRs are protected areas of land
and or coastal environments where in people are an integral component of the system. Together, they
constitute a worldwide network linked by international understanding for exchange of scientific
information.

Advantages of In-situ conservation:- The positive points of In-situ conservation over Ex-situ are:

 To ensure preservation of greater diversity of alleles and genotypes

 It permits continued natural evolution rather co-evolution with pests and diseases and natural
selection to occur, thus it sustains and generates diversity.

 For recalcitrant tree species, this is the only viable option at present

 It permits study of ecology of species, serves habitat protection/ ecosystem protection

objectives besides in many cases it saves regeneration and storage cost/space/ time

Ex-situ Conservation:-

Ex situ means away from the natural, original place or position. Ex situ conservation which
include conservation of plant and animals away from their natural habitat or ecosystem. Or Ex situ
conservation means literally, “off-site conservation”. It is the process of protecting an endangered
species of plants or animal outside of its natural habitat; for example, by removing part of the
population from a threatened habitat and placing it in a new location ,which may be a wide area or
within the care of humans.

If in situ conservation is not feasible due to various reasons, threatened species can only be
conserved ex situ (Jeffries, 1997; Wolf, 1999). Moreover, ex situ conservation serves as a source of
material for research and ecosystem restoration. However, ex situ conservation interrupts
evolutionary and ecological processes and limits genetic variability and adaptability of species to
changing environmental conditions.
 Ex-situ management is the simplest, cost-efficient approach that involves regular material
viability testing and timely recovery, depending on crop and reproductive systems. The Global Crop
Diversity Trust (GCDT) plays a key role in improving ex-situ conservation techniques and in
management of global crop diversity. Ex-situ conservation in the broad sense this form of
conservation can include the use of botanic gardens and arboreta on the one hand and gene bank on
the other.

Botanic gardens and arboreta represent the oldest forms of conservation in Europe ate back to
the 15th and 16th centuries. Little material has survived since then however apart form a few trees, so
that most are in fact of later origin.The conservation of components of biological diversity outside
their natural habitats often in a laboratory, collection in botanical garden, zoo or aquarium

The collections of seed banks of wildlife species are projected to play a key role for
preserving and restoring biodiversity. Therefore, it is necessary to efficiently manage the collections
of wild species for utilization and multiplication of adequate viable seeds. The seeds were divided
into two classes by Roberts as per their storage capacity in 1973.

 Orthodox seeds
 Recalcitrant seeds

Ex-situ

 In vitro: Base collection, seed stores, cryo preservation and Tissue culture Storage
 In vivo: Active collection (working collection) clonal repositories, Annual Field Gene Banks.

OR

 Seed Gene bank

 Field/Plant gene bank
 Meristem gene bank
 Cryopreservation
 DNA bank
 Botanical gardens etc.

Botanic gardens
 Botanic gardens are institutions holding documented collections of living plants for the
purposes of scientific research, conservation, display and education (Wyse Jackson, 1999). Botanic
gardens have played a vital role in the conservation of the world’s plant diversity. Many of the
world’s threatened plant species are represented in their living collections or seed banks which
collectively provide an insurance policy supporting the maintenance of global biodiversity (Waylen,
2006). In fact, botanic gardens have a strong focus on wild species which are endangered in their
natural habitat (Heywood, 1999; Wyse Jackson, 1998). In more recent years, some botanic gardens
started to accept new responsibilities and were designed to be broadly based botanical resource
centres (Wyse Jackson, 1998).

Botanic gardens worldwide maintain approximately 80,000 species as living plants (nearly
30% of the known vascular plant species of the world) represented by more than 4 million living
accessions (individual plant collections), and keep 250,000 seed bank accessions (Hawksworth,
1995; Wyse Jackson, 1999). Many botanic gardens around the world have developed effective
seedbanks for conserving germplasm of wild plant species. Botanic gardens, in cooperation with
other bodies, are increasingly involved in the in situ conservation of plant resources (Hawksworth,
1995; Heywood, 1999; Wyse Jackson and Sutherland, 2000; Bramwell, 2007).

Field genebanks

Field gene banks are important for conservation of plant species that do not produce seeds
and propagate vegetatively or produce the so-called recalcitrant seeds (seeds which cannot be stored
at low temperature). Usually these difficult-to-conserve species can be maintained as living
collections in field gene banks. On the other hand, conservation in field gene banks requires sound
information on the ecological requirements of the species in question. It needs area of sufficient size
and site conditions similar to that of the original population but with a lower environmental load or
pressure (Wolf, 1999).

Gene banks

Gene banks are important for the conservation of germplasm or genetic material. Germplasms
that can be stored in gene banks include seeds, pollen, spores, semen (sperms), eggs, embryos, cells
and tissues. More recently, DNA sequences are also being kept in specialized banks (Smith et al.,
2007). In any case, representative sampling is necessary. Approximately 6 million accessions of
plants are maintained in gene banks worldwide (Engels and Engelmann, 2002; Frison et al., 2002).
Germplasm conserved in gene banks serves for research and restoration purposes.

Cryopreservation

Cryopreservation is an important complementary ex situ tool to in situ conservation, as

genetic back-up in case of losses of genetic variation, and it is the strategy of choice when in situ
strategies are ineffective in avoiding excessive losses of genetic variation or breed extinction
(Gustavo et al,. 2007). The cryopreservation technique comprised vitrification of shoot apices
isolated from in vitro stock shoot cultures and somatic embryos.

Cryopreservation is a technique that ensures safe, long‐term conservation of genetic

resources of plant species with recalcitrant seeds, of vegetatively propagated species and of
biotechnology products such as somatic embryos, cell lines and genetically transformed material.
The technique was implemented at the end of the 20th century and could be used today for routine
cryo storage as long as some important factors were taken into consideration (Reed, 2001).

Tissue culture procedures are usually required to multiply super cooled material via axillary
shoots or somatic embryogenesis, and were improved for use with tree species in recent years (Nehra
et al. 2005). In addition, production of transgenic tree species and molecular breeding procedures
require functional cryopreservation protocols (Haggman et al. 2001).Three major genetic risks in ex
situ collections are genetic drift, adaptation to cultivation and mutation accumulation.

Germplasm maintenance:-

 Germplasm maintenance involves seed germination testing and monitoring the viability.
Germplasm samples for conservation are multiplied mainly during the post-rainy season to
get better quality seed.

 To minimize genetic drift, an adequate number of plants are grown and sampled equally in
constituting new seed stocks.

 Genetic integrity is maintained by pollination control while regenerating cross-pollinating

crops such as sorghum, pearl millet, and pigeonpea.
  Pre-storage deterioration in seed quality is minimized by harvesting promptly when maturity
is reached.

Maintaining seed viability:-

 Viability tests measure how many seeds germinate and develop into plants, which reproduce
themselves. Viability of accessions should be tested:

• Before seeds are packaged and placed in the gene bank, and

• At regular intervals during storage.

 Seed viability of conserved germplasm is tested at five years interval for medium-term
storage and at ten years interval for long-term storage

 Many methods are available to test seed-viability. The most accurate method to test seed
viability is the germination test using appropriate procedure.

Germination test

Complete germination can be achieved only under optimum conditions of light, temperature
and water. Sample size use a minimum of two replicates each of 50 or 100 seeds for testing initial
germination and two replicates each of 25 or 50 seeds for subsequent tests, depending on available
seed quantity, take a random sample of seeds from the container. If the seeds are very dry (moisture
content <8%) expose them to ambient atmosphere for 24 hr to raise the moisture content before
testing for germination. Two methods are used for testing germination:

1) Top of paper method for millets.

2) Between paper (Rolled towel) method for sorghum, chickpea, pigeonpea and groundnut.

Paper towel is used as substrate for germination in both these methods.

Topographical tetrazolium test for viability

The tetrazolium test can be used as a backup procedure to germination tests in gene banks. It
can be applied to firm seeds, which have failed to germinate at the end of germination test. The
tetrazolium test procedure includes the following steps:

1) Preconditioning :-
 Remove the seed covering structures (glumes, etc).
 Precondition the seeds by first soaking in water or by placing them on a moist
medium at 30°C.
2) Staining:-
 Bisect the seeds longitudinally through the embryo with a razor blade.
 Discard one-half of the seed and place the other half in the staining solution at
recommended concentration in a glass vial.
 Place the vials in an incubator maintained in the dark at recommended temperatures
and duration
 After staining, wash the seeds several times in distilled water to remove excess stain.
 Immerse the seeds in lactophenol (1 L of lactophenol prepared from 200 ml phenol,
200 ml lactic acid, 400 ml glycerine, and 200 ml water) solution for 1–2 h before
evaluation of the seeds.
 Evaluate the seeds for staining pattern under a low power binocular microscope.
 Viable tissues stain bright red. Pink and very dark red stains are indicative of dead
tissue.
 Classify the seeds into three categories depending on staining pattern:
  Completely stained and viable seeds,
 Completely unstained seeds that are nonviable, and
 Partially stained seeds.

Seed vigour tests:- Vigour is the sum total of all those properties in seed which upon sowing result
in rapid and uniform production of healthy seedlings under a wide range of environments, including
both favourable and stress conditions. Vigour tests supplement information about seed quality.

Seed health testing

Seed borne fungi such as Alternaria, Fusarium, Penicillium, Aspergillus and Rhizopus spp.
affect longevity during storage. Curators should ensure that seeds prepared for long-term
conservation are free from the seed borne pathogens. The methods employed to detect the pathogens
are referred to as seed health testing methods. The commonly used seed health testing methods are:-

 Visual examination Seeds

 Blotter test
 Agar plate method
 Seed health standard

Maintaining seed quantity:-

 Seed quantity in medium-term store is recorded at 5-years interval.

Regeneration of germplasm:-

Accessions are regenerated when seed viability and seed quantity are critical (<85% viability
and/or <1/4 of total quantity). Regeneration is carried out in the post rainy season for securing high
quality seed. Genetic integrity of accessions while regeneration in cross pollinated crops, sorghum,
pearl millet, pigeonpea is maintained by using different pollination control methods. Seed health tests
are carried out for regenerated germplasm. Species that do not produce seeds are maintained as live
plants in glasshouse or in field gene bank or in special facility of reinforced cement concrete (RCC)
rings.
Seed packing:- Seed packaging involves placing the dry seeds of an accession into a container for
storage. Seeds are packaged to:

 Prevent them from absorbing moisture from air.

 Avoid mix up of individual accessions.
 Prevent contamination by insects and diseases.
 Pack the seeds immediately after drying.
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