Ex situ conservation

Ex-situ Conservation
Submitted by: Kiran
(2019A46D)
Submitted to:
Dr. Somveer Nimbal

Contents
1. Components of ex-situ conservation
2. Plant genetic resources conservation in
gene banks, national gene banks and gene
repositories
3. Preservation of genetic materials under
natural conditions, Perma-frost conservation
4. Guidelines for sending seeds to network of
active/ working collections
5. Orthodox and recalcitrant seeds-
differences in handling
6. Clonal repositories
7. genetic stability under long term storage
condition.

Introduction
 Ex-situ conservation is the
preservation of components of
biological diversity outside their
natural habitats
 This involves conservation of genetic
resources (wild and cultivated species
 Such strategies include establishment
of botanical gardens, zoos,
conservation strands and gene, pollen
seed, seedling, tissue culture and

Components of Ex-situ
conservation
Ex-situ conservation
Gene banks
Field gene bank, Seed
gene bank, In-vitro bank,
DNA bank, Pollen bank
Botanical Garden

Gene banks
 It refers to a place or organisation
where germplasm to be conserved in
living state
 The most fundamental activity of gene
bank is to treat a new sample in a way
that will prolong its viability as long as
possible while ensuring its quality
 Plant samples must periodically be
grown out, regenerated and new
seeds harvested to maintain the
sample

Field gene banks
 Field gene banks also called plant gene banks
 Conservation of germplasm by growing in feild
 Those plant species that have recalcitrant seeds,
vegetatively propagated and are long lived perennials
conserved in field gene banks.
 In field gene banks, germplasm is maintained in the
form of plants as a permanent living collection.
 Field gene banks are often established to maintain
working collections of living plants for experimental
purposes. They are used as source of germplasm for
species such as coconut, rubber, mango, cassava, yam
and cocoa. Field gene banks have been established in
many countries for different crops.

Horticulture Crop
Field gene bank
Field gene bank in Phillippines

Advantages Disadvantages
Suitable for species with
recalcitrant and intermediate
seeds
Vulnerable to changes in
management practices
Convinient fr
characterisation and
evaluation
Susceptible to pests,
diseases and other natural
or human driven calamities
such as drought, neglect
and war
Easily accesible for use Limited amount of genetic
diversity conserved
Possible to combine
conservation and research
High maintainance costs,
not suitable for long term

Seed Gene Banks
 Germplasm is conserved in form of seeds
 The largest seed bank in world is the Millennium Seed
Bank housed at the Wellcome Trust Millennium
Building (WTMB), located in the grounds of Wakehurst
Place in West Sussex, near London
 Advantage: allows large population to be preserved
and minimises the genetic erosion by providing
optimum conditions and reduces the need for
regeneration

In-vitro Gene Bank
 Conservation of PGR in the form of small parts
economizes the space and allows conserving
vegetatively propagated an medium and long term
storage conditions.
 Two major techniques in In vitro storage:
a) In vitro Bank: In this technique, buds, protocorm
and meristematic cells are preserved through
particular light and temperature arrangements in a
nutrient medium. This technique is used to
preserve seedless plants and plants that
reproduce sexually.
b) Cryobank: In this technique, a seed or embryo is
preserved at very low temperatures. It is usually
preserved in liquid nitrogen at -196 °C. This is
helpful for the conservation of species facing
extinction.

DNA Bank
 DNA can be stored as extracted uncut genomic DNA
or may be maintained in genomic library on bacteria
or virus.
 Storage is performed by immobilisation of the DNA
onto nylon membrane.
 DNA bank is a particular type of genetic resource
bank that preserves and distribute the DNA molecule
and provides associated informationAdvantages Disadvantages
Minimum space required for storage Not a practical germplasm conservation
method
Applicable to all plant species Well established facilities and skills is
required
Effective and rapid method
Large number of sample can be
handled

Pollen Bank
 'Pollen Banks' are used to store pollen
grains for a short as well as very long
period of time in a viable conditions.
Advantages Disadvantages
More effective in case of
recalcitrant species
It may lead to loss of organelle
genome, thus require effective
plant generation protocol
Minimum space required for
storage
Tricellular pollen storage
extremely difficult
Intra nad inter population
variation can be conserved
Propagules not readily available
Only half of the genome

Purpose of Gene Banks
 Physical facilities for maintaining collections of live plant
materials – domesticated cultivated plants – wild plant
species (crop wild relatives and other wild plant species
useful for food and agriculture and other end uses) –
entire plants, seeds, pollen, embryos, meristems, cells,
or DNA, depending on the biology of the species
 It is a facility for maintaining crop diversity .
 Usually this diversity is in the form of seeds, stored and
conserved in a frozen state
 Some gene banks use normal household freezers for
this purpose
 The ideal temperature is between -10and -20
 Each different type is stored in its own container
 Such as a bottle, a can or a sealed aluminum foil
package

 Maintaining material in genebanks is often
termed ‘ex situ conservation’ – defined as ‘the
conservation of components of biodiversity
outside their natural habitats’ (CBD, 1992)
 Whereas in situ conservation is maintenance
of viable population in their natural
surroundings – a dynamic system which
allows the biological resources to evolve and
change over time through natural selection
processes
 Both concepts are therefore fundamentally
different but are complementary.

Activities in Gene Bank
 Collecting and Acquisition – assembling the
collection
 Processing – assessing the quantity, viability,
health of samples and preparation for storage
 Storage – in a cold store, laboratory or in the field
 Regeneration and Multiplication – periodically
rejuvenating and increasing the material
 Characterization and Evaluation
 Documentation, Inventory – maintaining and
making available detailed records on each
sample
 Distribution – of clean, disease-free seeds, or
other planting material, to requestors

National Gene banks for major crops
CROP CENTRE
Wheat IIWBR, Karnal
Rice NRRI, Cuttack
Potato CPRI, Shimla
Cotton CICR, Nagpur
Sugarcane SBI, Coimbatore
Tobacco CTRI, Rajahmundry
Pulses IIPR, Kanpur
Tuber crops( except potato) CTCRI, Trivandrum
Plantation crop CPCRI, Kasorgod
Oilseed crop IIOr, Hyderabad
Horticulture crop IIHR, Bangalore
Soybean DOSR, Indore
Groundnut DOGR,Junagarh
Maize IARI, New Delhi

Botanical gardens
 A botanical garden is a place where plants,
especially ferns, conifers and flowering plants, are
grown and displayed for the purposes of research
and education
 The botanical garden in Nashik is the first of its kind in
the country and only one in South East Asia with a
unique laser show.
 Botanical garden Guru Nanak Dev University, Amritsar
Punjab Botanical garden Punjabi University, Patiala
Punjab and National Cactus and Succulent Botanical
Garden and Research Centre in Punchkula (Haryana)
 There are other botanical garden like NBRI, Lukhnow,
Indian Botanical Garden, Kolkata, IARI,Delhi, FRI,
Dehradun and Royal Botanical garden, Kew England
 There are over 2500 botanical gardens in 148
countries

Permafrost
 Permafrost is any ground that remains completely
frozen—32°F (0°C) or colder—for at least two years
straight. These permanently frozen grounds are most
common in regions with high mountains and in Earth’s
higher latitudes—near the North and South Poles
 Permafrost is made of a combination of soil, rocks and
sand that are held together by ice. The soil and ice in
permafrost stay frozen all year long
 These are the places for storage of germplasm without
involving high cost in refrigeration
 Eg. Genebank located in Svalbard Seed Vault in the
island of Spitsbergen. Samples are stored in glass
ampules and moisture content is around 2-4%.
Thetemperature in the mine is constant from -2 to 3⁰C.

Permafrost in india
Doomsday vault present at 17,500 m
high on a cliff top in Himalayas,
Chang-La has sub zero temeprature
and low humidity necessary to
suspend seed life for future
generation.

Working Collection
Active Collection
Base Collection
Orthodox seeds
Recalcitrant seeds
Classification of seed
banks

 Base collections: Seeds can be conserved under long term
(50 to 100 years), at about -20⁰C with 5% moisture
content. They are disturbed only for regeneration.
 Active collection: Seeds are stored at 0 ⁰ C temperature
and the seed moisture is between 5 and 8%. The storage is
for medium duration, i.e., 10-15 years. These collections are
used for evaluation, multiplication, and distribution of the
accessions.
 Working collections: Seeds are stored for 3-5 years at 5-
10 ⁰C and the usually contain about 10% moisture. Such
materials are regularly used in crop improvement
programmes.
 Orthodox seeds: Seeds which can be dried to low
moisture content and stored at low temperature without
losing their viability for long periods of time is known as
orthodox seeds. (eg.) Seeds of corn, wheat, rice, carrot,
papaya, pepper, chickpea, cotton, sunflower.
 Recalcitrant: Seeds which show very drastic loss in viability
with a decrease in moisture content below 12 to 13% are
known as recalcitrant seeds. (e.g) citrus, cocoa, coffee,
rubber, oilpalm, mango, jack fruit etc.

Guidelines for sending seeds to
network of active/working collections
 Plant germplasm proposed to be registered should be submitted to
NBPGR
 Material is to be accompanied with properly filled Form –A (Annexure
I) duly signed by the applicant and Head of the institution with official
rubber seal (15 copies, each attached documentary evidences
submitted).
 Form A accompanied complete description of the germplasm material
using standard descriptors (as per concerned crop AICRP or NBPGR
descriptors). Include photograph(s) of plant/plant parts/crop and /or
fingerprints (DNA or biochemical profile), if available.
 A declaration to the effect that working-stock for supply to users would
be maintained by the instiution associated with the development of the
material. It may be ensured by the Director/PD/PC or Competent
Authority of the concerned organization.
 Another declaration is that such germplasm does contain any gene or
gene sequence involving terminator technology would also be

Guidelines for submitting the orthodox
seed material
 Seed material dried to low moisture level without loss of seed viability.
 Minimum number of 4000 seeds in case of cross-pollinated crop species,
2000 in self –pollinated and 100-1000 in difficult species, such as some
vegetables, wild relatives etc. submitted.
 Seed supplied from a fresh harvest and should not be more than 60 days
old.
 Seeds supplied sound, healthy, physiologically mature and collected from
healthy plants
 For providing good quality healthy seeds. It is advised to dry the seed
material in shade immediately after the harvest.
 The potential viability of seeds should be more than 85% in most crop
species except in special cases, such as cotton, forage grasses,
sugarcane some vegetable crops etc.
 Seeds should not be treated with chemicals.
 Seeds packed in good quality paper, muslin cloth or plastic packet(s) with
proper identity.
 If required, the packets should be packed in card-board boxes to
minimize damage and moisture absorption.

Guidelines for Submission of
Recalcitrant / Intermediate Seed
MaterialThe guidelines to be follows are given below:
 Preferably, more than 1000 seeds supplied. However, recognizing the
importance of material, even small quantity may be acceptable. Supply
of additional seeds may help develop DNA profiles.
 To avoid any injury to the fruit surface they should be sent in aerated
polythene bags/cardboard boxes in the form of complete fruit.
 If fruits are bulky and difficult to transport, the seeds should be
extracted without causing any injury and should be transported within
48 hrs, packed in saw dust/charcoal /peat moss etc.
 Avoid transporting at high temperature (above 30⁰C). Store and
transport should be preferably in moist conditions between 15-20⁰C
temperature conditions.
 Extracted seeds should be treated with suitable fungicide (0.1 %
Captan or Thiram powder).
 Avoid air drying and washing of seeds.
 In remaining cases the genetic material should be supplied to relevant
NAGS in the form of propagules establishment in the field gene bank
following the guidelines given below.

Guidelines for Submission of
Propagules
 10-25 propagules (depending on crop) supplied to the concerned
NAGS( Nationa Active Germplasm Site) for their maintenance in
field repository or in-vitro repository (if available) with a request
for an acknowledgement.
 Concerned NAGS should be informed in advance about the
supply of material to facilitate processing and establishment of
germplasm.
 Genetic material, stocks, propagules of non-orthodox seed
producing crops are generally being maintained in the form of
grafts, slips, propagules, seedlings and plants.
While supplying genetic material following steps and precautions
are to be followed depending on the crop:
 The slips, grafts, propagules or plants supplied to the NAGS
should be free from insects, weeds and diseases as far as
possible. The material should be welllabelled and packed
properly in aerated polythene bags. During the dry summer the
grafts of crafts should be wrapped in moist moss grass to retain
the moisture.

 In case of crops like coconut, the material should be
sent either as embryos or seedlings. If the embryos
need to be transferred from the field, the embryos
embedded in the endosperm should be packed in
the sterile plastic bag with sterile moist cotton. These
should be kept in the refrigerator overnight and
transferred in the same box with proper labels on it.
 In case of seedlings the embryos should be grown
using the river sand in plastic bags/boxes. Once the
seedlings are established these should be
transferred to bigger pots. The healthy, vigorous
seedlings should be supplied.
 The material should be packed in small
wooden/card-board boxes with proper aeration. Also,
these boxes should be well marked with labels at 3
or 4 places “To be handled carefully: seedlings’’ in
order to avoid any damage during transit.
 The material should be sent to the NAGS
immediately after harvest. To avoid any delay in
transfer, use speed post or reliable courier services

Drying and storage:
 Drying to equilibrium in a controlled environment of 5-20°C and 10-25 % of relative
humidity, depending upon species
 Storage - in air-tight container for long term storage
 Long-term conditions (base collections); temperature of -18 ± 3°C , relative
humidity of 15% ± 3
 For medium-term conditions (active collection) samples are stored under
refrigeration at 5-10 °C and relative humidity of 15 % ± 3% – medium-term storage
conditions are adequate for 30 years.
Seed viability Monitoring: Timing
 After cleaning and drying or at the latest within 12 months after receipt of the
sample
 200 seeds for initial germination tests (ISTA, 2008) followed by sequential testing,
if the initial germination is less than 90 % (Ellis et al. 1985) during storage
 100 or smaller seed samples If there are not sufficient seeds; conducted with
replications.
 Viability: Variability- initial germination threshold - above 85% for most cultivated
crop species; lower value acceptable for specific accessions, wild and forest
species
Regeration:
 The viability drops below 85 % of the initial viability
 The remaining seed quantity is less than what is required for three sowings of
representative population of the accession. The most-original-sample to be used
 In practice when accession does not have sufficient seeds for long-term storage
(e.g. 1500 seeds for a self-pollinating species and 3000 for an out-crossing
species)
 Deposit in long-term storage for reference purposes - at least 50 seeds of the

 Assessment of water content, vigour and viability:
1. The storage category of the seed should be determined immediately
by assessing its response to dehydration.
2. The water content should be determined individually, on separate
components of the propagule, and in a sufficient number of plants.
3. The vigour and viability should be assessed by means of germination
tests and in a sufficient number of individuals.
4. During experimentation, cleaned seed samples should be stored
under conditions that do not allow any dehydration or hydration.
 As a first step to preservation, it is important to ascertain the seed
storage category by assessing the response of the propagule to
dehydration.
 The response to drying will in turn determine the treatment needed for
cryostorage.
 The rate and uniformity of germination of a seed sample, or of seed-
derived explants, is a reliable indicator of vigour, while the totality of
germination (i.e. what proportion/percentage of seeds or explants tested
finally germinated) reveals the overall viability of the sample.
 Viability should not be not less than 80% in a sample.
 The recommended temperatures for tropical/sub-tropical species and
those of temperate origin are 25 °C and 15 °C respectively (Pritchard et

Hydrated storage of recalcitrant seeds
 Hydrated storage, and is achieved by holding the seeds in a
closed under saturated relative humidity (RH) conditions.
 Hydrated storage should be carried out under saturated RH
conditions, and seeds should be maintained in air-tight
containers, at the lowest temperature that they will tolerate
without damage
 All seeds should be decontaminated prior to hydrated storage
and infected material should be eliminated.
 Stored seeds must be inspected and sampled periodically to
check if any fungal or bacterial contamination has occurred, and
whether there has been any decline in water content and/or
vigour and viability.
 The basic principle for maximising the storage life span of
recalcitrant seeds is that water contents should be retained
at essentially the same levels as those characterising the
newly-harvested state. Thus the seeds must not lose water
either before or after being placed in storage.
 Ideally sealing polythene bags with an inner paper bag inside
(‘bag within a bag’) or sealing plastic buckets of appropriate size
for the seed numbers, are favoured for storage (Pasquini et al.
2011).

 Storage temperatures should be the lowest that seeds of
individual species will tolerate, without any deleterious effect on
vigour and viability
 For recalcitrant seeds of temperate origin, temperatures of 6 ± 2
°C are generally suitable for storage, while for the majority of
seeds of tropical/subtropical origin, 16 ± 2 °C is normal range
 Under hydrated storage conditions, fungi (or less frequently
bacteria) are likely to proliferate, so vigilance and appropriate
action to curtail seed-to-seed infection is required. If infected
seeds are not removed they will contaminate the entire batch in
a storage container. This renders the stored seeds useless and
eliminates their potential for supplying explants for
cryopreservation
 Depending on the duration of hydrated storage, containers
should be briefly and periodically ventilated to avoid
development of anoxic conditions at which time the contents of
containers must be inspected and any contaminated seeds
discarded.
 Storing seeds in a monolayer is ideal, but if seeds are stored in
several layers, the seeds should be mixed about during
aeration.

 Clonal repositories contain active collections that hold and
propagate agriculturally important germplasm, such as
strawberries, raspberries, fruit trees, coffee and nuts that for a
variety of reasons are not usually held in active collections as
seed.
 The primary responsibilities of the repositories are to collect,
identify, propagate, preserve, evaluate, document and distribute
clonal germplasm as part of the NPGS. This includes
maintenance of an information file on each accession in the
clonal collection.
 The repositories are charged with developing active global
collections of appropriate wild species and domestic cultivars
and to assemble a maximum level of genetic diversity possible
for each genus and species for which they are responsible.
 They also conduct research to improve evaluation, propagation,
characterization and preservation of clonal germplasm.
 The national clonal germplasm repositories are intended to
carryout by the regional stations. Unlike seeds held at the

 Many clonal crops can be conserved as seed, but they are impossible
to maintain true to type by raising plants from seed.
 Many clonally propagated species take a long time to mature, and
they are best preserved as mature live plants for plant breeding and
research.
 Clonal collections are expensive to establish and they have many of
the same problems that confront seed collections.
 Accessions must be maintained as plants in the field , which can
require large tract of land or in screen houses or green houses.
 Accessions may also be maintained as lives sticks of budwood held
under refrigeration or as tissue cultures.
 There may be many losses during maintenance from insects and
disease, freezing temperatures, electric power failures and grazing
animals.
 Clonal preservation is more expensive and labour intensive than seed
storage.
 Clonal collections have been threatened as facility or land use
priorities have been changed.
 By establishing the national clonal repositories, a mechanism for
stable, longterm maintenance for many important clonally propagated
species has been provided

 “Clonal stability depends on the
method of preservation, the length of
time, and the inherent genetic stability
of the clone
 Mutations can occur as a result of
background gamma- and X-ray
irradiation, exposure to mutagenic
chemicals, and other environmental
influences.
 Each clone has its own natural rate of
mutation, so generalizations cannot be
made.” Westwood (1989)

 Monitor genetic stability: Genetic shifts in
the collection can arise from genetic instability
of accessions due to chromosomal changes
and gene mutations causing morphological or
biochemical variation. These genetic
instabilities are manifested as somaclonal
variation in tissue culture.
 Avoid propagating off-types: Individuals of a
clone should be inspected regularly for off-
types. If the accession appears to be a mixture
of genotypes, characterization data should be
used to determine which is the correct
propagule. Herbarium specimens or
photographs of accessions may also be useful
for verifying the identity of questionable
specimens.

 Accessions from which seeds are collected for
propagation (grasses and legumes) can lose
genetic information over time if too few
individuals of an accession are maintained and
self pollination occurs in a percentage of
cases. This problem is lessened in accessions
that primarily produce seed apomictically.
Species that inter cross easily lose superior
genotypes when cross-pollination is not
controlled and seeds are used.
 Studies on seed physiology and reproductive
biology would identify grasses that could be
stored as bulked seed and would reduce the
size of field collections. Crops of this type
need to establish protocols for maintaining
accessions according to breeding behaviour
(Sackville Hamilton and Chorlton 1997).

Evaluate genetic stability:
 Monitoring collections for genetic stability is not an easy task.
 Each crop has specific descriptors that can be used to
characterize the accessions.
 A well-identified collection may be used to develop RNA or DNA
fingerprinting, which may be used for future evaluations.
 The relevance of the existing techniques is discussed in a
publication entitled ‘Using Molecular Marker Technology in
Studies on Plant Genetic Diversity’ (de Vicente and Fulton
2003).
Avoid mixing propagules:
 Mixing can easily occur in fields where roots, stolons, rhizomes
and runners can invade adjacent plots.
 Repotting or planting errors and growth of propagules such as
runners into adjacent pots may occur in screenhouses and
greenhouses.
 Wide spacing of pots can be used to limit growth of stolons or
runners into adjacent pots.
 Careful attention by staff members will minimize planting errors.

Minimize labelling and handling mistakes:
 More than one identification number (i.e.
plot number and accession number) should
be used when planting or harvesting fields.
 A field map should be available showing the
sequence of planting, and, if possible,
labels should be printed from computer
files, or carefully checked to minimize the
problem.
 Labels should be indelible and as
indestructible as possible.
 Advance planning before any planting or
repotting effort will minimize errors.

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