Germplasm conservation refers to maintaining plant genetic material, such as seeds or living plants, in a way that minimizes the risk of loss. This allows the material to be used in the future if needed. There are two main approaches: in-situ conservation keeps germplasm in its natural habitat through methods like biosphere reserves and national parks, while ex-situ conservation stores germplasm outside its natural habitat using techniques like seed banks, field gene banks, and botanical gardens. The goal of both is to preserve genetic diversity and protect endangered plant species and economically important varieties.

1. GERMPLASM
CONSERVATION
AND
CRYOPRESERVATION
SAKEENA ASMI T
MAHATMA GANDHI
UNIVERSITY

2. GERMPLASM?
Germplasm broadly refers to the hereditary
material (total content of genes) transmitted to
the offspring through germ cells.
In other words, germplasm refers to a whole
library of different alleles of a species.
Germplasm contains the information for a species‘
genetic makeup, a valuable natural resource of
plant diversity.
Germplasm provides the raw material for the
breeder to develop various crops.
For plants, the germplasm may be stored as a seed
collection(even a large seed bank).
For trees, in a nursery.

3. WHY CONSERVATION?
• Until two decades ago the genetic resources were
getting depleted owing to the continuous
depredation by man.
• Great diversity of plants are needed to keep the
various natural ecosystems functioning stably.
• Humans are dependent on plant species for food
and many different uses. E.g: Basic food crops,
building materials, oils, lubricants, rubber and
other latexes, resins, waxes, perfumes, dyes fibres
and medicines.
• Aesthetic value of natural ecosystems and the
diversity of plant species are also important.

4. Conti…
• It was imperative therefore that many of the elite,
economically important and endangered species
are preserved to make them available when
needed.
• The conventional methods of storage failed to
prevent losses caused due to various reasons.
LIKE………

5. 1. ATTACK BY PEST AND PATHOGENS
2. CLIMATE DISORDER
3. NATURAL DISASTERS
4. POLITICAL AND ECONOMIC CAUSES
The conventional methods of germplasm
preservation are prone to possible
catastrophic losses because of:
• So new methodologies have been devised for
long term preservation of materials.

6. WHAT IS GERMPLASM
CONSERVATION?
• Germplasm conservation refers to maintain the collected
germplasm in such a state that there is minimum risk for
its loss and that either it can be planted directly in the
field or it can be prepared for planting with relative ease
when ever necessary.
• The very objective of germplasm conservation (or storage)
is to preserve the genetic diversity of a particular plant or
genetic stock for its use at any time in future.
• In recent years, many new plant species with desired and
improved characteristics have started replacing the
primitive and conventionally used agricultural plants.
• It is important to conserve the endangered plants or else
some of the valuable genetic traits present in the primitive
plants may be lost.

7. GERMPLASM CONSERVATION

8. There are basically two approaches for germplam
conservation of plant genetic materials:
1. In-situ conservation
2. Ex-situ conservation

10. In-situ conservation
• Conservation of germplasm under natural habitat is
referred to as in situ conservation.
• In-situ means in the natural origin, place or position.
• It involves conservation of plants and animals in their
native ecosystems.
• In situ conservation is the preservation of species and
populations of living organisms in a natural state in the
habitat where they naturally occur.
It includes : 1. BIOSPHERE RESERVES
2. NATIONAL PARKS
3. GENE SANCTUARIES
4. ON FARM CONSERVATION

11. BIOSPHERE RESERVES
• A biosphere reserve is an area proposed by its habitats,
ratified by a national committee and designated by
UNESCO’s Man and Biosphere(MAB) program in 1971,
which demonstrates innovative approaches to living and
working in harmony with nature.
• The term “Biosphere” refers to All the land, water and
atmosphere that supply life on earth.
• The word “reserve” means that it is a special area
recognised for balancing conservation with sustainable use.
• Each biosphere reserve demonstrates practical approarches
to balancing conservation and human use of an area.

12. BIOSPHERES IN INDIA

14. NATIONAL PARKS
• National park may be defined as an area , declared by
state, for the purpose of protecting, propagating or
developing wildlife there in, or its natural environment for
their scientific ,educational and recreational value.
• India's first national park was established in 1936 as Hailey
National Park, now known as Jim Corbett National Park,
Uttarakhand.
• By 1970, India only had five national parks.
• As of July 2015, there were 103 national parks
encompassing an area of 40,500 km2 (15,600 sq ),
comprising 1.23% of India's total surface area.

16. JIM CORBETT NATIONAL PARK SUNDARBAN NATIONAL PARK
KAZIRANGA NATIONAL PARK

17. GENE SANCTAURY
• A gene sanctuary is an area where plants are conserved.
• India has set up its first gene sanctuary in the Garo Hills
of Meghalaya for wild relatives of citrus.
• Efforts are also being made to set up gene sanctuaries
for banana, sugarcane, rice and mango. The genetic
diversity is sometimes conserved under natural habitat

18. GARO HILLS, MEGHALAYA

19. ON-FARM CONSERVATION
• On-farm conservation is defined as “A form of in situ
conservation in the place where the domesticated or
cultivated species have developed their distinctive
properties.”
• The maintenance of domesticates such as landraces or
local crop varieties in farmers fields often referred to as
on-farm conservation (Maxted et al. 2002), in agro or
‘inter situ’ (Blixt 1994).
• There is an urgent need to also pay attention to the
many economically important wild species that are
neither on farm nor in protected areas.

21. ADVANTAGES OF IN-SITU
CONSERVATION
• Plants are conserved in their natural environment.
• Biodiversity permanently protected.
• Representative examples of ecosystems also permanently
protected.
• Natural and cultural heritage protected permanently.
• Ecological integrity is maintained and managed.
• Opportunities may arise for ecologically sustainable land
uses. (which come with associated economic benefits)
• Facilitates scientific research of the site.
• It may be possible to improve the ecological integrity of
the area and restore it if it has been damaged by
poaching etc.

22. DISADVANTAGES OF IN-SITU
CONSERVATION
• Endangered habitats may be fragmented so the area
may not be large enough to ensure the survival of these
species.
• Genetic diversity may have already been dramatically
decreased.
• Conditions that threatened the organisms in the area
may still be present, e.g. disease or interspecific
competition.
• Poachers and ecotourists may see the thriving area as an
opportunity and may cause damage.

23. 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 future use.

24. TYPES OF EX SITU
CONSERVATION
GENE BANK
• Gene bank refers to a place or organization
where germplasm can be conserved in living
state.
• Gene banks are mainly three types:-
1. SEED GENE BANK :
A place where germplasm is conserved in
the form of seeds is called seed bank.
2. FIELD GENE BANK :
Field gene banks also called plant gene
bank are area of land in which germplasm
collections of growing plants are assembled.

25. 3. MERISTEM GENE BANK :
Germplasm of asexually propagating
species can be conserved in the form
of meristems. It is used in conservation
of horticultural crops.
Conti…
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.
POLLEN BANK - pollen can be preserved
limited space. Pollen preservation may be
useful for base collections of species that
do not produce orthodox seeds.

26. Botanical garden –
• There are more than 1,700 botanical gardens and
institutions worldwide holding plant collections that
serve both conservation and educational purposes.
• Botanical garden and zoos are the most conventional
methods of ex situ conservation like NBRI, Lucknow,
Acharya Jagadish Chandra Bose Indian Botanical Garden
Kolkata, IARI Dehli, FRI, Dehradun, Royal botanical Garden,
Kew, England

27. NBRI, Lucknow
Acharya Jagadish
Chandra Bose Indian
Botanical Garden,
Kolkata

28. Royal Botanical Garden,
Kew, England

30. WHAT IS ARTIFICIAL SEED..?
• Artificial seed can be defined as artificial encapsulation
of somatic embryos, shoot bud or aggregates of cell of
any tissues which has the ability to form a plant in in-vitro
or ex-vivo condition.
• Artificial seed have also been often referred to as synthetic
seed.

31. • Artificial seeds were first introduced in 1970’s as a novel analogue
to the plant seeds.
• The production of artificial seeds is useful for plants which do not
produce viable seeds. It represents a method to propagate these
plants.
• Artificial seeds are small sized and these provides further advantages
in storage, handling and shipping.
• The term, “EMBLING” is used for the plants originated from
synthetic seed.
• The use of synthetic varieties for commercial cultivation was first
suggested in Maize (Hays & Garber, 1919).
HISTORY

32. 1. DESICCATED SYNTHETIC SEEDS
Desiccated synthetic seeds are produced nacked or
polyoxyethylene glycol encapsulated somatic embryos. This
type of synthetic seeds is produced in desiccation tolerant
species plant.
2. HYDRATED SYNTHETIC SEEDS
Hydrated synthetic seeds are produced by encapsulating
the somatic embryos in hydrogels like sodium alginate,
potassium alginate, carrageenan, sodium pectate or sodium
alginate with gelatine.
BASED ON THE TECHNIQUES TWO
TYPES OF ARTIFICIAL SEEDS ARE
PRODUCED

33. • Development of artificial seed production technology is currently
considered as an effective and efficient method of propagation in
several commercially important agronomic and horticultural crops.
• These artificial seed would also be a channel for new plant lines
produced through biotechnological advances to be delivered directly
to greenhouse and field.
• High volume propagation potential of somatic embryos combined
with formation of synthetic seeds for low-cost delivery would open
new ways for clonal propagation in several commercially
important crop species.
NEED FOR ARTIFICIAL PRODUCTION
TECHNOLOGY

34. BASIC REQUIREMENT FOR THE
PRODUCTION OF ARTIFICIAL SEEDS.
• One pre-requisite for the application of synthetic seed technology in
micropropagation is the production of high quality,
1. Vigorous Somatic Embryos that can produce plants with frequencies
comparable to natural seeds.
2. Inexpensive production of large numbers of high quality somatic
embryos with synchronous maturation.
3. Encapsulation and coating systems, though important for delivery
of somatic embryos, are not the limiting factors for the development
of synthetic seeds.
4. Commercialization of synthetic seeds.

35. Establish somatic embryogenesis
Mature somatic embryogenesis
Synchronize and singulate somatic
embryos
Mass production of somatic embryos
Standardization of encapsulation
Standardization of artificial endosperm
Mass production of artificial seeds
Greenhouse and field planting
PROCEDURE FOR PRODUCTION OF
ARTIFICIAL SEEDS

36. Methods for artificial seed
encapsulation
1. Dropping method
• Somatic embryos are dipped in hydrogel, this step encapsulate SEs.
• Hydrogel used may be any of the following.
o Alginate – sodium alginate, agar from see weeds, seed gums like
guar gum, locust bean gum.
- Sodium alginate solution (1 – 5%), prepared in MS basal medium
solution.
- SEs are dipped in this solution.
- These coated beads are added one by one into a complexation
solution flask kept on magnetic stirrer and kept such for around
20-30 minutes.

37. • Embryos get covered by calcium alginate which is a stable complex
due to ionic bond formation, become harder, seeds become harder.
• Then gelled embryos are washed with water or MS basal medium.
• The synthetic seeds are ready.
2. Molding method
• This method follows simple procedure of mixing of embryos with
temperature dependent gel (e.g. gel rite, agar).
• Cells get coated with the gel at lowering of the temperature.

39. POTENTIAL USES OF ARTIFICIAL SEEDS
DELIVERY SYSTEMS:
 Reduced costs of transplants.
 Direct greenhouse and field delivery of :
- elite, select genotypes.
- hand-pollinated hybrids.
- genetically engineered plants.
- sterile and unstable genotypes.
 Large-scale mono cultures.
 Mixed-genotype plantations.
 Carriers for adjuvants such as microorganisms, plant growth
regulators, pesticides, fungicides, nutrients and antibiotics.
 Protection of meiotically-unstable, elite genotypes.
 Can be conceivably handled as seed using conventional planting
equipment.

40. POTENTIAL USES OF ARTIFICIAL SEEDS
ANALYTICAL TOOLS:
 Comparative aid for zygotic embryogeny.
 Production of large numbers of identical embryos.
 Determination of role of endosperm in embryo development and
germination.
 Study of seed coat formation.
 Study of somoclonal variation.

41. ADVANTAGES OF EX-SITU
CONSERVATION
• It is possible to preserve entire genetic diversity of crop
species at one place.
• Less interaction with environment, so less chance to loss
of genetic resources.
• Ex situ conservation removes targets from their natural
habitat and conserves them in a seed bank, botanical
garden etc.
• Handling germplasm is easy.
• Small propagules stored in less space, no need to
generate plants every season.
• The germplasm preserved can be maintained in an
environment free from pathogens.
• It can be protected against the natural hazard.

42. DISADVANTAGES OF EX-SITU
CONSERVATION
• Monitoring of viability frequently is essential in seed
gene banks.
• High cost of maintenance.
• Some plants do not produce fertile seeds.
• Loss of seed viability.
• Seed destruction by pests, etc.
• Poor germination rate.
• This is only useful for seed propagating plants.
• It’s a costly process.
• Conditions that threatened the organisms in the area may
still be present, e.g. disease or interspecific competition.

43. IN VITRO STORAGE

44. Various methods of in-vitro
conservation
1. Cryopreservation - generally involves storage in liquid
nitrogen.
2. Cold storage - it involves storage in low and non
freezing temperature.
3. Low pressure – it involves partially reducing the
atmospheric pressure of surrounding.
4. Low oxygen storage - it involves reducing the oxygen
level but maintaining the pressure.

45. CRYOPRESERVATION
• Cryo is a Greek word (krayos – frost).
• It literally means preservation in “frozen state.”
• Cryopreservation is the technique of freezing cells and
tissues at very low temperatures (sub-zero temperature,
typically -196oC) at which the biological material remains
genetically stable and metabolically inert, while
minimizing ice crystal formation.
• Any biological activity including the biochemical reactions
that would cause cell death are effectively stopped.

46. HISTORY
• Ernest John Christopher Polge, an English biologist, was
the first person to solve the mystery of how to preserve
living cells at very low temperatures in 1949. He acciden
tly discovered the cryoprotective properties of glycerol
on fowl sperm.
• During early 1950s, James E. Lovelock suggested that
increasing salt concentrations in a cell as it dehydrates
to lose water to the external ice might cause damage to
the cell. He also proposed that the mechanism of action
of cryoprotectants.
• In 1953, the research work of Jerome K. Sherman led
him to successfully freeze and thaw human sperm.
• In 1983, Alan Trounson, was credited for successfully
achieving a pregnancy after freezing early human
embryos one to three days after fertilization.

47. Principle :
• To bring plant cells or tissue to a zero metabolism and
non dividing state by reducing the temperature in the
presence of cryoprotectant.
• Cryopreservation is a non-lethal storage of biological
material at ultra-low temperature. At the temperature of
liquid nitrogen (-196 degree) almost all metabolic
activities of cells are ceased and the sample can then
be preserved in such state for extended peroids.
• However, only few biological materials can be frozen
to (-196 degree) without affecting the cell viability.

48. It can be done :
1. Over solid carbon dioxide (at -79 degree)
2. Low temperature deep freezer (at -80 degree )
3. In vapor phase nitrogen (at -150 degree)
4. In liquid nitrogen (at -196 degree)

49. Why Liquid nitrogen ?
1. Chemically inert
2. Relatively low cost
3. Non toxic
4. Non flammable
5. Readily available
 Liquid nitrogen is most widely used material for
cryopreservation.
 Dry ice can also be used.

50. 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.

51. STEPS INVOLVED IN
CRYOPRESERVATION
1. SELECTION OF PLANT
MATERIAL
2. PREGROWTH
3. ADDITION OF
CRYOPROTECTANTS
4. VITRIFICATION
5. CRYOPROTECTIVE
DEHYDRATION

52. Conti…
6. ENCAPSULATION AND
DEHYDRATION
7. FREEZING
8. STORAGE
9. THAWING
10. DETERMINATION OF
SURVIVAL OR VIABILITY

53. 1. SELECTION OF PLANT
MATERIAL
• Morphological and physiological conditions of plant
material influence the ability of explants to survive
during cryopreservation.
• Different types of tissues can be used for
cryopreservation such as:
 Ovules
 Anther/pollen
 Embryos
 Endosperm
 Protoplast etc.

54. FACTORS:
o Tissue must be selected from healthy plants.
o Small & young.
o Rich in cytoplasm.
o Meristematic cells can survive better than the larger.
o Highly vacuolated cells.

55. o Callus derived from tropical plant is more resistant to
freezing damage.
o A rapidly growing stage of callus shortly after 1 or 2
weeks of subculture is best for cryopreservation.
o Old cells at the top of callus and blackened area should
be avoided.
o Cultured cells are not ideal for freezing. Instead,
organized structures such as shoots apices, embryos or
young plantlets are preferred.
o Water content of cell or tissue used for cryopreservation
should be low freezable water, tissues can withstand
extremely low temperatures.

56. 2. PREGROWTH
• Pregrowth treatment protect the plant tissues against
exposure to liquid nitrogen.
• Pregrowth involves the application of additives known
to enhance plant stress tolerance.
E.g :
 Abscisic acid
 Proline
 Trehalose

57. 3. ADDITION OF
CRYOPROTECTANTS
• A cryoprotectant is a substance that is used to protect
biological tissue from freezing & thawing damage
(damage due to ice formation).
• They acts like antifreeze.
• They lower freezing temperature.
• Increase viscosity and
• Prevents damage to the cells.

59. Penetrating cryoprotectants
• Penetrating cryoprotectants are so called because they
penetrate the cell membrane and enter the cytosol.
• They are exclusively small molecules.
• They form hydrogen bonds with water to prevent ice
crystallisation.
• They act by replacing water and therefore controlling
cell size changes as well as preventing intracellular ice
formation and prevent excessive dehydration during cell
cryopreservation.
• Common penetrating cryoprotectants are DMSO-
(Dimethyl sulfoxide), glycerol, ethylene glycol.

60. Non-Penetrating cryoprotectants
• This type of cryoprotectants do not penetrate the cell
membrane.
• They are larger molecules, usually polymers such as
polyethylene glycol or saccharides such as sucrose.
• Non-penetrating cryoprotectants are thought to act by
dehydrating the cell before freezing, thereby reducing the
amount of water that the cell needs to lose to remain
close to osmotic equilibrium during freezing.
• They inhibit ice growth by the same mechanism as
penetrating cryoprotectants, but they do not enter cells.
• They help to dehydrate the cell prior to cryopreservation
by altering the osmotic balance and also help to prevent
damage to cells during recovery from cryopreservation
by preventing solutes, particularly larger protoplasmic
elements, from escaping the cell too rapidly.

61. There are several cryoprotectant
which include (DMSO), glycerol,
ethylene, propylene , sucrose,
mannose, glucose , proline and
acetamide. Among these DMSO,
sucrose and glycerol are most
widely used.

62. 4. VITRIFICATION
• The term “vitrification” refers to any process resulting in
“glass formation”, the transformation from a liquid to a
solid in the absence of crystallization.
• According to this definition, cells that are properly slow
frozen become “vitrified”.
• A process where ice formation cannot take place because
the aqueous solution is too concentrated to permit ice
crystal nucleation. Instead, water solidifies into an
amorphous ‘glassy’ state.

63. 5. CRYOPROTECTIVE DEHYDRATION
• Dehydration can be achieved by growth in presence of
high concentration of osmotically active compounds like :
 Sugars
 polyols and/or
 In a sterile flow cabinet
 over silica gel.

64. 6. ENCAPSULATION AND
DEHYDRATION
• This involves the encapsulation of tissues in calcium
alginate beads.
• Which are pre-grown in liquid culture media containing
high concentration of sucrose.
• After these treatments the tissues are able to withstand
exposure to liquid nitrogen without application of
chemical cryoprotectants.

65. 7. FREEZING
• After addition of cryoprotectants, freezing is done in
such a way that it does not cause intracellular freezing
& crystal formation.
• The following types of freezing is done in the process of
cryopreservation :
1. Rapid Freezing
2. Slow Freezing
3. Stepwise Freezing

67. Ampoules used during freezing FREEZERS
FREEZING OF
CELLS

68. 8. STORAGE
• Storage of frozen material at correct temperature is as
important as freezing.
• The frozen cells/tissues are kept for storage at
temperature ranging from -70 to -196°c.
• Temperature should be sufficiently low for long term
storage of cells to stop all the metabolic activities and
prevent biochemical injury.

69. 9. THAWING
• It is done by putting ampoule containing the sample in a
warm water bath (35 to 40°c).
• Frozen tips of the sample in tubes or ampoules are
plunged into the warm water with a vigorous swirling
action just to the point of ice disappearance.
• It is important for the survival of the tissue that the
tubes should not be left in the warm water bath after ice
melts .
• Just a point of thawing quickly transfer the tubes to a
water bath maintained at room temperature and
continue the swirling action for 15 sec to cool the warm
walls of the tube.
• Tissue which has been frozen by encapsulation/dehydration
is frequently thawed at ambient temperature.

70. 10. DETERMINATION OF
SURVIVAL/VIABILITY
• Regrowth of the plants from stored tissues or cells is the
only test of survival of plant materials.
• Various viability tests include Fluorescien diacetate(FDA)
staining, growth measurement by cell number, dry and
fresh weight.
• Important staining methods are:
 Triphenyl Tetrazolium Chloride (TTC)
 Evan’s blue staining.

72. Advantages
1. Once the material is successfully conserved to particular
temperature it can be preserved indefinitely.
2. Once in storage no chance of new contamination of
fungus or bacteria.
3. Minimal space required.
4. Minimal labour required.

73. Applications
• It is ideal method for long term conservation of genetic
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.
• Biodiversity conservation.
• Conservation of plant germplasm.