The document discusses germplasm conservation, including both ex situ and in situ methods. Ex situ conservation involves maintaining genetic resources outside their natural habitat, such as in seed banks, field gene banks, DNA banks, botanical gardens, and through in vitro and cryopreservation methods. In situ conservation preserves species in their natural environments through biosphere reserves, national parks, wildlife sanctuaries, and on-farm conservation. Cryopreservation is described as a method to bring plant cells and tissues to a zero metabolism state through freezing at very low temperatures in liquid nitrogen.

1. Conservation and Preservation of
Germplasm
Lecture #: 2
By
Miss Andleeb Tajammal
University of Gujrat

2. Germplasm Conservation
 The germplasm has to be maintained in such a state
that there is minimum risk for its loss
• Plant germplasm is genetic source material in the
form of seeds, cultured cells, pollens, callus
• The in situ / ex-situ preservation of these material
is known as “Germplasm Conservation’’

3. Factors that Effect Genetic Recourses !
• Deforestation
• Urbanization
• Pollution
• Habitat destruction
• Fragmentation and degradation
• Climate change
• Globalization
• Market economies
• Over-grazing and changes in
land-use pattern are contributing
indirectly to the loss of diversity

4.  To improve the effectiveness of sustainable management
and conservation of biodiversity through adequate
conservation, use and handling of genetic resources.
 Conservation of plant genetic resources is necessary for
food security and agro-biodiversity
 Biodiversity provides a valuable source of compounds to
the medical, food and crop protection industries.
 Genetic diversity provides options to develop through
selection and breeding of new and more productive
crops, resistant to biological and environmental stresses.
Why Conservation?

6. Types of Conservation
• Short term (Working Collections)
(>3-5 years) at 10-15 ℃ at 10% moisture
• Medium Term (Active Collections)
10-15 year at temperature 15 ℃ 5% moisture
• Long Term (Base Collections)
50 years more stored about -20 ℃ with 5%
moisture content

7. Ex Situ Conservation
• Ex situ means away from the natural, original place
or position. Ex situ conservation which include
conservation of plants and animal away from their
native ecosystem
Ex situ conservation is the conservation of genes or
plant genotypes outside their environment of natural
occurrence, for current or for future use.

8. Types of Ex Situ Conservation
1) Gene Bank
• Gene bank refers to a place or organization where
germplasm can be conserved in living state
• Gene banks are mainly three types
a) Seed Gene bank
b) Field Gene bank
c) Meristem Gene bank
 Seed Gene Bank
• A place where germplasm is conserved in the form of
seeds is called seed bank

9.  Field Gene Bank
• Field gene banks also called plant gene bank are area
of land in which germplasm collections of growing
plants are assembled
• It is also called ex-situ conservation of germplasm
 Meristem Gene Bank
• Germ plasm of asexually propagating species can be
conserved in the form of meristems
• It is used in conservation of horticultural crops

10. 2) DNA Bank
• Where DNA can be stored as extracted uncut genomic DNA
Such efforts have lead to storage of total genomic
information of germplasm in the form of DNA libraries
3) Pollen Bank
• Pollen can be preserved in limited space
• Pollen preservation may be useful for base collections of
species that do not produce orthodox seeds
4) Botanical Garden
• Globally more than 1,700 botanical gardens and institutions
holding plant collections that serve both conservation and
educational purposes
• Botanical garden and zoos are the conventional methods of
ex situ conservation like,
• Lahore Botanical Garden,
• Quaid-i-Azam University Botanical
Garden, Islamabad,
• Royal botanical Garden, kew England

11. In Situ Conservation
• In situ conservation is the preservation of species and
populations of living organisms in a natural state in
the habitat where they naturally occur
• Types of In situ Conservation
1) Biosphere Reserves
2) National Parks
3) On Farm Conservation
4) Wild Life Sanctuaries

12. 1) Biosphere Reserves
Biosphere reserves or natural reserves are multipurpose
protected areas with boundaries circumscribed by
legislation.
 The main aim of biosphere reserve is:
 To preserve genetic diversity in representative
ecosystems by protecting wild animals, traditional life
style of inhabitant and domesticated plant/animal genetic
resources.

13. • The concept of Biosphere Reserves was launched in
1975 as a, part of UNESCO’s ‘Man and Biosphere
Programme, dealing with the conservation of
ecosystems and the genetic resources contained
therein.
• Biospheres Reserves in Pakistan
1. Lal Suhanra, 1977
2. Ziarat Juniper Forest, 2013

14. 2. National Parks
• National parks defined as:
• “An area , declared by state, for the purpose
of protecting, propagating or developing
wildlife there in, or its natural environment
for their scienctific, educational and
recreational value’’

15. • Deosai National Park
• Chitral National Park
• Khunjerab National Park

16. 3. Wildlife Sanctuary
• Wildlife sanctuaries are areas dedicated to the
protection and maintenance of wildlife
• These areas are protected and managed for a variety
of reasons including:
 Scientific research
 Wilderness protection
 Preservation of species and Genetic diversity
 Maintenance of environmental services
 Protection of specific natural and cultural
features
 Tourism and sustainable use of natural
resources

17. 4. On-farm conservation
“A form of in situ conservation in the place where the
domesticated or cultivated species have developed their
distinctive properties”
Maintenance of
domesticates such as
landraces or local crop
varieties in farmers fields
often referred to as on-farm
conservation

19. In Vitro Method For Germplasm
Conservation
• In vitro conservation is the conservation of tissues,
cells, genomes and genes under controlled
environmental conditions
• In vitro method employing shoots, meristems and
embryos are ideally suited for the conservation of
germplasm

20. Advantages
• Large quantities of material
can be preserved in small
space
• Germplasm preserved can
be maintained in an
environment free from
pathogens
• It can be protected against
the nature’s hazards
• From the germplasm stock
large number of plants can
be obtained whenever
needed

21. Methods For the In Vitro Conservation of
Germplasm
 Cropreservation
• generally involves storage in liquid nitrogen
 Cold storage
• it involves storage in low and non freezing
temperature.
 Low –pressure
• involves partially reducing the atmospheric
pressure of surrounding
 low oxygen storage
• it involves reducing the oxygen level but
maintaining the pressure

22. Cryopreservation
“Cryopreservation (Greek,krayos-frost) literally means
in the frozen state’’
 The principle involved in cryopreservation is to bring the plant
cells and tissue cultures to a zero metabolism or non-dividing state
by reducing the temperature in the presence of cryoprotectants
 Cryopreservation broadly means the storage
of germplasm at very low temperature
• Over solid carbon dioxide (at 79°C)
• Low temperature deep freezer (at -80°C)
• In liquid nitrogen (at -196°C)

23. Mechanism of Cryopreservation
• The technique of freeze preservation is based on the
transfer of water present in the cells from a liquid to solid
state
• Due to the presence of salts and organic molecules in the
cells, the cell water requires much more lower
temperature to freeze(even up to -68°C) compared to the
freezing point of pure water (around 0°C)
• When stored at low temperature, the metabolic processes
and biological deteriorations in the cells/tissues almost
come to standstill

24. Mechanism of cryopreservation
involves following steps
• Selection of material
• Addition of cryoprotectant
• Freezing
• Storage in liquid nitrogen
• Thawing
• Washing and reculturing
• Measurement of viability
• Regeneration of plants

26. 1. Selection of Material
• Selection of plant species and the tissue with
particular references to the morphological and
physiological characters largely influences the ability
of the explants to survive in cryopreservation.
• Any tissue from a plant can be used for
cryopreservation
 For example,
Meristems,embryos,endosperm,ovules,
seeds, culture plants.

27. 2. Addition of Cryoprotectant
• Cryoprotectant are the compound that can prevent the
damage caused to cells by freezing or thawing
• There are several cryoprotectants which include:
• Dmso
• Glycerol
• Ethylene
• Propylene Sucrose
• Mannose
• Glucose

28. 3.Freezing
• Sensitivity of the cells to low temperature is
visible and largely depends on the plant species
• Four different types of freezing are used
1. Slow freezing method
2. Rapid freezing method
3. Stepwise freezing method
4. Dry freezing method

29. 4. Storage
• Maintenance of the frozen cultures at the specific
temperature is as important as freezing
• Frozen cells/tissues are kept for storage at
temperature in the range of -72 to-196°C
• Storage is ideally done in liquid nitrogen refrigerator
at 150°C in the vapour phase or at -196°C in the
liquid phase

30. 5. Thawing
• Carried out by plunging the frozen sample in ampoules into the
warm water (temp 35-45°C) bath with vigorous swirling
• By this approach, rapid thawing (at the rate of 500- 750°C min-
1) occurs, and this protects the cell from the damaging
• As the thawing occurs (ice completely melts) the ampoules are
quickly transferred to a water bath at temperature 20-25°C

31. 6. Reculture
• In general thawed germplasm is washed
several times to remove cryoprotectant
• Material is then cultured in a fresh media

32. 7. Plant Regeneration
• Purpose of cryopreservation of germplasm is to
regenerate the desired plant for appropriate plant
growth and regeneration, the cryopreserved cell /
tissue have to be carefully nursed and grown
• Addition of certain growth promoting substances,
besides maintenance of appropriate environmental
conditions often necessary for successful plant
regeneration

33. Applications
• Ideal method for long term conservation of
material
• Disease free plants can be conserved and
propagated
• Recalcitrant seeds can be maintained for long time
• Endangered species can be maintained
• Pollens can be maintained to increase longitivity
• Rare germplasm and other genetic manipulations
can be stored