This document provides information on in vitro germplasm conservation. It discusses that germplasm conservation aims to preserve the genetic diversity of plants. There are several methods of in vitro conservation including cryopreservation, cold storage, and low pressure/low oxygen storage. Cryopreservation involves freezing plant cells and tissues at ultra-low temperatures like in liquid nitrogen to bring their metabolism to zero. It allows for long term conservation of large amounts of genetic material in a small space. Cold storage conserves germplasm at low non-freezing temperatures to slow growth. Low pressure and low oxygen storage reduce atmospheric pressure and oxygen concentration to inhibit plant tissue growth.

1. IN-VITRO GERMPLASM
CONSERVATION
PREPARED BY: KALPATARU NANDA
I.D NO. : 03PBG/16

2. WHAT IS GERMPLASM ?
The sum total of all the genes present in a crop and its related
species constitutes its germplasm. It is ordinarily represented by a
collection of various strains and species.
Plant germplasm is the genetic source material used by the plant
breeders to develop new cultivars.
They may include:
 Seeds
Other plant propagules (Which can be grown into mature plant) are:
 Leaf
 Stem
 Pollen
 Cultured cells
Germplasm provides the raw material (genes) which the breeder
used to develop commercial crop varieties. Therefore, germplasm is
the basic indispensable ingredient of all breeding programmes. Thus a
great emphasis is placed on collection, evaluation and conservation of
germplasm.

3.  The conservation of germplasm involves the preservation of the
genetic diversity of a particular plant or genetic stock. It can be used at
any time in future.
 It is important to conserve the endangered plants or else some of the
valuable genetic traits present in the existing and primitive plants will be
lost.
 Some crops produce recalcitrant or short lived seeds.
 Similarly, in case of clonal crops seeds are not the best material to
conserve due to their genetic heterogeneity and unknown worth. Their
genes need to be conserved.
 The roots and tubers loose viability rapidly. Their storage requires large
space, low temperature and is expensive. In addition, materials
modified by genetic engineering may some, times be unstable. Such
materials are needed to be conserved intact for future .A global
organization- International Board of Plant Genetic
Resources (IBPGR) has been established for germplasm conservation
and provides necessary support for collection, conservation and
utilization of plant genetic resources through out the world.
APPLICATIONS OR SIGNIFICANCE OF
GERMPLASM CONSERVATION

4. IN VITRO METHOD FOR GERMPLASM CONSERVATION
 In vitro method employing shoots, meristems and embryos are
ideally suited for the conservation of germplasm.The plant with
recalcitrant seeds and genetically engineered can also be
preserved by this in vitro approach.
 There are several advantages associated with in vitro
germplasm conservation:-
 Large quantities of material can be preserved in small space.
 The 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.

5. THERE ARE THREE MAIN APPROACHES FOR THE
IN VITRO CONSERVATION OF GERMPLASM
 CRYOPRESERVATION
 COLD STORAGE
 LOW –PRESSURE AND LOW OXYGEN –STORAGE
Other approaches:-
 Slow Growth Cultures
 Desiccated Somatic Embryos (SE) and Artificial Seeds
 DNA Clones

6. IN VITRO CONSERVATION OF GERMPLASM..

7. CRYOPRESERVATION
Cryopreservation (Greek,krayos-frost)literally means in the
frozen state. The principle involved in cryopreservation to bring
the plant cells and tissue cultures to a zero metabolism or
non-dividing state by reducing the temperature in the
presences of cryoprotectants (DMSO (dimethyl sulfoxide),
glycerol, ethylene, propylene, sucrose, mannose, glucose,
praline, acetamide etc ).
CRYOPRESERVATION broadly means the storage of germplasm at
very low temperature using :-
 Over solid carbon dioxide(at 79°C)
 Low temperature deep freezer(at -80°C)
 Using vapour nitrogen (at- 150°C)
 In liquid nitrogen( at -196°C)

8.  Among these, the most commonly used cryopreservation
is by employing liquid nitrogen. At the temperature of
liquid nitrogen(at -196°C), the cell stay in a completely
inactive state and thus can be conserved for longer period.
Infact cryopreservation has been successfully applied for
germplasm conservation of some plant species e.g
rice,wheat,peanut,sugarcane,coconut.
 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.
MECHANISM OF CRYOPRESERVATION

10. TECHNIQUE OF CRYOPRESERVATION
The cryopreservation of plant cell culture followed the
regeneration of plants broadly involves the following
stages.
 Development of sterile tissue culture.
 Addition of cryoprotectant and pretreatment.
 Freezing
 Storage
 Thawing
 Reculture
 Measurement of survival/viability
 Plant regeneration

11. 1.DEVELOPMENT OF STERILE TISSUE CULTURE
The 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
e.g.meristems,embryos,endosperm,ovules,seeds,culture
plants.
2.ADDITION OF CRYOPROTECTANT
Cryoprotectant are the compound that can prevent the
damage caused to cells by freezing or thawing.There
are several cryoprotectant which include:
(DMSO, GLYCEROL, ETHYLENE, PROPYLENE,
SUCROSE, MANNOSE, GLUCOSE…..)

12. 3.FREEZING
The sensitivity of the cells to low temperature is visible and
largely depends on the plant species.Four different types of
freezing are used.
 Slow freezing method
 Rapid freezing method
 Stepwise freezing method
 Dry freezing method
4.STORAGE
Maintenance of the frozen cultures at the specific temperature is
as important as freezing.In general,the 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.
The ultimate objective of storage is to stop all the cellular
metabolic activities and maintain their viability .For long term
storage temperatue at -196°C in liquid nitrogen is ideal.

13. LOOK OF STORAGE TECHNIQUE!

14. 5.THAWING
Thawing is usually 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°Cmin-1)occurs, and this
protects the cell from the damaging effects ice crystal
formation.
As the thawing occurs (ice completely melts) the ampoules
are quickly transferred to a water bath at temperature 20-
25°C. This transfer is necessary since the cells get
damaged if left for long in warm(35-45°C) water bath.
6.RECULTURE
In general thawed germplasm is washed several times to
remove cryoprotectant. The material is then cultured in a
fresh media.

15. 7.PLANT REGENERATION
The ultimate 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 conditionis often
necessary for successful plant regeneration.

16. COLD STORAGE
 Cold storage is a slow growth germplasm conservation
method. It conserves the germplasm at a low and non-
freezing temperature (1- 9°C). The growth of the plant
material is slowed down in cold storage in contrast to
complete stoppage in cryopreservation. Thus it prevents
cryogenic injuries. Long term cold storage is simple, cost
effective. It yields germplasm with good survival rate. Virus
free strawberry plants could be preserved at 10°C for about
6 years. Several grape plants have been stored for over 15
years by using a cold storage at temperature around 9°C
and transferring them in the fresh medium every year.

17. LOW PRESSURE AND LOW OXYGEN STORAGE
 In low- pressure storage, the atmospheric pressure
surrounding the plant material is reduced. In the low
oxygen storage, the oxygen concentration is reduced. The
lowered partial pressure reduces the in vitro growth of
plants. In the low-oxygen storage, the oxygen
concentration is reduced and the partial pressure of
oxygen below 50 mmHg reduces plant tissue growth.
Due to the reduced availability of 02, and reduced
production of CO2, the photosynthetic activity is reduced. It
inhibits the plant tissue growth and dimension. This method
has also helped in increasing the shelf life of many fruits,
vegetables and flowers.

18. SLOW GROWTH CULTURES
 Slow-growth of plantlets in-vitro provides an attractive
alternative to freeze preservation of germplasm as it is
simpler, cheaper and very effective. Slow growth may be
achieved by maintaining the plantlets either at a low
temperature (4-9°C or Ca. I5°C) or on a medium having
high osmotic concentration (e.g., 20% sorbitol or
sucrose) or both.
 In addition, the nutritional status of the medium may be
lowered to restrict the growth of plantlets. Under the
conditions of slow-growth, cultures may be attended to only
once in several months. Its subculture may, be necessary
only after long periods, once every 236 months.

19.  The slow-growth approach is being utilized for germplasm
conservation of specified root, tuber and tree species by the
NBPGR, New Delhi. A National Facility for Plant Tissue
Culture Repository has been created for this purpose. It has
so far developed the slow-growth protocols for ginger,
garlic, banana, sweet potato, etc
 The techniques for desiccation of SE s and for production
of desiccated, artificial seeds are now becoming available.
The desiccated SEs and artificial seeds can be stored at
low (4°C) or ultralow (-20°C) temperatures for prolonged
periods in a manner similar to zygotic seeds..
Desiccated Somatic Embryos (SE) and
Artificial Seeds

20. DNA CLONES
 Germplasm can also be conserved in form of DNA
segments cloned in a suitable vector, e.g., cosmids,
phasmids or YACs. The technique is highly
sophisticated, technically demanding and expensive.
 It is likely to be used for conservation of valuable .genes
or DNA segments from threatened species. It can also be
used for the conservation of the entire genomes of
various germplasm lines of different species.

21. LIMITATION OF IN-VITRO GERMPLASM
CONSERVATION
 The expensive equipment needed to provide controlled
and varible rates of cooling/warming temperatures can
however be a limitation in the application of in vitro
technology for large scale germplasm conservation.
 Formation of ice crystal inside the cell should be
prevented as they cause injury to the cell.
 Sometimes certain solutes from the cell leak out during
freezing.
 Cryoprotectant also effect the viability of cells.

22. STORAGE STABILITY USING CRYOPRESERVATION: A CASE STUDY IN
PAPAYA [2011]
Ashmore, S.E.Drew, R.A.Kaity, A.
Abstract
 Ex situ conservation of Carica papaya and its crop wild relatives has been the subject of ten
years of research in our laboratory. This paper summarises the achievements and key
findings of this work. Both clonal and seed materials have been investigated to allow storage
of elite clonal material as well as maximum genetic diversity. Clonal material from several
genotypes has been successfully stored using micropropagation followed by shoot tip
cryopreservation, with >70% regeneration after 12 months storage in LN. The
regeneration and acclimatisation of cryopreserved plants has been achieved for field testing
and observations of morphological characteristics, including flowering and fruiting, have not
detected any changes associated with cryopreservation. Genetic stability following shoot tip
cryopreservation has also been monitored using both RAF (Random Amplified DNA
Fingerprinting) and AMP (Amplified DNA Methylation Polymorphism) techniques. Whilst both
RAF and AMP changes were observed following cryopreservation, these were not associated
with cryopreservation per se and did not correlate with any modification in plant morphology.
Investigations of papaya seed storage indicate that seed is essentially orthodox, but
dormancy breaking treatments were required for germination. However, germination post
storage for 12 months was >68% at ultra-low temperatures (LN), but <5% at
conventional seed storage temperatures (-20°C). This is in line with recent evidence
that cryopreservation enhances storage stability and longevity, even in orthodox seeds,
when compared with standard seed bank approaches. This highlights the importance of
cryopreservation for the long-term management of genetic resources of both clonal and seed
genetic resources of papaya.

23. CRYOPRESERVATION OF ORCHID GENETIC RESOURCES BY DESICCATION:
A CASE STUDY OF BLETILLA FORMOSANA
Rung‐Yi Wu, Shao‐Yu Chang, Ting‐Fang Hsieh, Keng‐Chang Chuang,IeTing, Yen‐Hsu Lai
and Yu‐Sen Chang
Abstract
 Many native orchid populations declined yearly due to climate change.
This resulted in some wild orchids being threatened. In order to maintain
the orchid genetic resources, development of proper methods for the
long-term preservation is urgent. Low temperature or dry storage
methods for the preservation of orchid genetic resources have been
implemented but are not effective in maintaining high viability of certain
orchids for long periods. Cryopreservation is one of the most acceptable
methods for long-term conservation of plant germplasm. Orchid seeds
and pollens are ideal materials for long-term preservation (seed
banking) in liquid nitrogen (LN) as the seeds and pollens are
minute, enabling the storage of many hundreds & thousands of
seeds or pollens in a small vial, and as most species germinate
readily, making the technique very economical. This article describes
cryopreservation of orchid genetic resources by desiccation and a case
study of Bletilla formosana.

24. THANK YOU………….