CRYOPRESERVATION

(ACTIVITY)

ABSTRACT
Cryopreservation—storing seeds in ultra-cold liquid nitrogen—is one method for

maintaining plant genetic stocks in seed banks. Can seeds withstand a really deep freeze

and still germinate?

OBJECTIVE

Investigate how freezing seeds for different amounts of time affects their

germination rate.

INTRODUCTION

Scientists have been using cryopreservation to build a "living" archive of seed

genes. Some of these frozen seeds are kept in storage in the event of natural disasters

that may wipe out an important crop. The question remains about seed viability and

how long seeds can remain in a frozen state and still germinate to produce a healthy

plant. In this science fair project, you will investigate how freezing seeds affects their

ability to germinate.

TERMS AND CONCEPT

1. You should understand the difference between these two areas of

study: cryopreservation and cryobiology.

2. You will need to understand seed structure and germination to understand what

impact cryopreservation will have on the seed.

3. You should also understand exactly what happens to plant tissues when seeds

are frozen. What is the process for freezing plant tissue for preservation? What

are some of the disadvantages/risks of this process?

MATERIALS AND EQUIPMENT

  Seeds; we suggest using the following seeds for this experiment (at least 5 of

each type):

o Supersweet corn

o Onion

o Lima bean

o Cabbage, radish, broccoli (choose one)

o Lettuce

 Access to a lab with liquid nitrogen

 Petri dishes, available from Carolina Biological, item #: 741250

 Cotton rolls, available from Carolina Biological, item #: 712635

 Water

 Oven

EXPERIMENTAL PROCEDURE

1. Suspend the seeds in liquid nitrogen. You should test at least four different time

durations in the liquid nitrogen. You should also test at least five of each of the

seed types in each of your experiments. Sometime duration suggestions:

o Experiment 1: 1 day

o Experiment 2: 2 days

o Experiment 3: 3 days

o Experiment 4: 4 days

2. After the appropriate length of time, remove the seeds from the liquid nitrogen.
 3. Prepare the petri dishes for germination. Cut the cotton slightly smaller than the

diameter of the petri dish. Saturate the cotton in the petri dish with water and

allow the dish to drain for 5 minutes as excess water will literally drown the

seeds.

4. Place the seeds in the petri dish and place them in the oven at about 70°–80° F.

5. Observe and record the rate of germination for each of the seeds every 24 hours

until they are ready for planting.

6. Which time duration in the liquid nitrogen led to the best germination results?

BIBLIOGRAPHY

What is cryobiology and cryopreservation:

Wikipedia Contributors. (2012, February 15). Cryopreservation. Wikipedia: The Free

Encyclopedia. Retrieved November 14, 2014, from
http://en.wikipedia.org/wiki/Cryopreservation

Wolfe, J. and Bryant, G. (1999). Cryobiology and anhydrobiology of cells. The University
of New South Wales. Retrieved February 28, 2012, from
http://www.phys.unsw.edu.au/~jw/cryoblurb.html

Visit this website to see time-lapse plant videos showing germination and other plant
activities:

Hangarter, R. (n.d.). Corn Germination. Plants-In-Motion. Retrieved February 28, 2012,

from
http://plantsinmotion.bio.indiana.edu/plantmotion/earlygrowth/germination/germ.htm
l

A book offering in depth coverage of research in cryopreservation in plants, including

developments in methods for specific species of plants:

Towill, L.E. 2002. Cryopreservation Of Plant Germplasm: Introduction And Some

Observations. Pp 3-21. In L.E. Towill And Y.P.S. Bajaj (EDS.)CRYOPRESERVATION Of Plant
Germplasm Ii. Biotechnology In Agricultural And Forestry Series Vol 50. Springer, London.
 CRYOPRESERVATION
(Report)
HISTORY

 Theoreticians of cryopreservation was James Lovelock (born 1919).

Osmotic stress
— Salt concentration
 Christopher Polge, carried out cryopreservation of first fowl sperm.
 Later, in 1950s they tried it on humans where pregnancy was obtained after
insemination of frozen sperm.
 However, the rapid immersion of the samples in liquid nitrogen did not produce
the necessary viability to make them usable after thawing.
 Importance of controlled or slow cooling to obtain maximum survival on thawing
of the living cells.
 Cryoprotectants were introduced.

CRYOPRESERVATION DEFINATION
Cryo is Greek word. (krayos – frost)

It literally means preservation in “frozen state.”

The principle - to bring plant cells or tissue to a zero metabolism and non dividing
state by reducing the temperature in the presence of cryoprotectant.

It can be done :

 Over solid carbon dioxide (at -79 degree)

 Low temperature deep freezer (at -80 degree )
 In vapor phase nitrogen (at -150 degree)
 In liquid nitrogen (at -196 degree)

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
periods.

However, only few biological materials can be frozen to (-196 degree) without affecting
the cell viability.

Liquid nitrogen- is most widely used material for cryopreservation.

Why Liquid nitrogen?

 Chemically inert
 Relatively low cost
 Non toxic
 Non flammable
 Readily available

CRYOPRESERVATION DEFINATION (Cont…)

• The use of very low temperatures to structurally preserve intact living

cells and tissue

• Unprotected freezing is normally lethal to cells while controlled cooling

can be used to produce stable conditions that preserve life

BENEFITS OF CRYOPRESERVATION

• Generation of safety stocks

• Saves time and money

• Preservation of cells

• Insurance against phenotypic drift

• Standard for experiments

Why preservation is important?

 Until two decades ago the genetic resources were getting depleted owing to the
continuous depredation by man.
 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.
 A new methodology had to be devised for long term preservation of material.

Major advantages are:

1. Once the material is sucessfully conserved to particular temperature it can be
preserved indefinately.

2. Once in storage no chance of new contamination of fungus or bacteria.

3. Minimal space required.

4. Minimal labour required

MECHANISM OF CRYOPRESERVATION
The cryopreservation technique followed by the regeneration of plants involves
following steps:

1. Selection of plant material:

Selection of proper plant material is important.

Two important factors depend on it such as (a) nature and (b)density.
Any tissue can be selected for this purpose. e.g. meristem, embryo, ovules, seeds
etc..
The density should be high.

2. Addition of Cryoprotectant

 They are chemical which prevent cryo destruction.

 These are sucrose, alcohols, glycols, some amino acid (proline), DMSO (dimethyl
sulfoxide).
 Generally two cryoprotectant should be used together instead of single one as
they are more effective.

Cryoprotectants
• Dimethyl sulfoxide (DMSO) and glycerol are the two most widely used
cryoprotectants
• Aid in preserving cells
• Encourage dehydration
• Minimize solution effects

3. Freezing

The sensitivity of cells to low temperature depends on the plant species.

There are four different types of methods :
 Slow freezing method - In this method the rate of freezing is slow (0.1-
10°C / min). It is commonly used for animal germplasm. • In this due to
cooling below freezing point, extra cellular crystals are formed not
intracellular.
 Rapid freezing method - This method is simple & easy. • Freezing is done
quickly so that there should be least change or development of
intracellular crystals.
 Stepwise freezing method- In this temperature gets lowered by -20°C to
-40°C & allows protective freezing of the cells. • Freezing stopped for 30
mins & then rapidly freezed in liq.N ; this results in decline in
temperature & good results are obtained.
4. Storage
 The maintenance of the frozen cells or material at specific temperature is very
important.
In general the temperature is kept -70 to -196 degree.
Prolong storage is done at temperature of -196 degree in liquid nitrogen.
To prevent damage, continuous supply of nitrogen is done.

5. Thawing
Usually carried out by plunging the vials into warm water bath with vigorous
swirling.
As thawing occurs the vials are transferred to another bath at 0 degree

6. Washing and Reculturing

The preserved material is washed few times to remove the cryoprotectant.
This material is then recultured in a fresh medium.

7. Measurement of viability
There is possibility of death of cells due to storage stress.
Thus viability can be found at any stage.
It is calculated by formula : No of cells growing / no of cells thawed * 100

8. Plant regeneration
The viable seeds are cultured on non-specific growth medium.
Suitable environmental conditions are maintained.
APPLICATIONS OF CRYOPRESERVATION

EMBRYO CRYOPRESERVATION
• It is used for embryo storage when in-vitro fertilization has resulted in more
embryo than is currently needed.
• Pregnancies have been reported from embryos stored for 16 yrs.
• The result has been uniformly positive with no increase of birth defects or
developmental abnormalities.

OOCYTES CRYOPRESEVATION
• Human oocytes cryopreservation is a new technology in which a woman’s
eggs(oocytes) are extracted, frozen & stored.
• Later, when a woman is ready to become pregnant, the eggs can be thawed,
fertilized & transferred to the uterus as embryos.

SEMEN CRYOPRESERVATION
• Semen can be used successfully & indefinitely after cryopreservation.
• The longest reported successful storage is 22 yrs.
• It can be used for sperm donation where recipient wants the treatment in a
different time or place or as a means of preserving fertility.

TESTICULAR CRYOPRESERAVTION
• Cryopreservation of immature testicular tissue is a developing method to avail
reproduction to young male who need to have gonadotoxic therapy.
• Health offsprings have been obtained after transplantation of frozen testicular
cell suspension or tissue pieces.

CRYOPRESERVATION EQUIPMENT
 Laboratory Freezer

 Ln2 Controlled Rate Freeze

 Glacier G50
  Blast freezer

References:
https://www.kean.edu/~evassili/images/Cryopreservation%20Chpt.%2020.ppt

https://www.atcc.org/~/media/PDFs/webinars/Presentations/2016/ATCC
%20Webinar%20Cryopreservation.ashx

https://s3.amazonaws.com/ppt-download/cryopreservation-151023075253-
lva1-app6891.pdf?response-content-disposition

Republic of the Philippines

SULTAN KUDARAT STATE UNIVERSITY
Graduate School
ACCESS, EJC Montilla, Tacurong City

Biological
Instrumentation
Final Requirement

Prepared by:
Mariel S. Bermudez
 MAT-Science

Submitted to:
Annie Francisco,MS
Professor