Cryopreservation is the process of preserving living cells and tissues at very low temperatures, typically in liquid nitrogen at -196°C. It works by slowing metabolism to a halt, preventing biological deterioration. Successful cryopreservation requires preventing ice crystal formation inside cells through cryoprotectants and controlling the freezing and thawing process. Organogenesis is the formation of plant organs directly from explants or indirectly through a callus phase, guided by growth regulators like auxins and cytokinins. It involves dedifferentiation of explant cells into a callus through rapid cell division, followed by redifferentiation forming organ primordia.

1. Introduction to Cryo and
Organogenic Differentiation
Submitted to: Miss. Shruti Gupta
Submitted by: Hrishika Sharma
B.Sc (Hons) Biotechnology IIIrd yr
[ Plant Biotechnology ]

2. Contents
• Introduction to Cryo
>Principle
>Mechanism
>Precaution/Limitation
>Techniques
• Organogenic Differentiation
>Types of Organogenesis
>Stages

3. Introduction to Cryopreservation
• The word cryo comes from the Greek word "karyos" meaning "frost".
It means preservation in a "frozen state".
• It is the process of cooling and storing cells, tissues, or organs at very
low temperature .
• It is a technique in which low temperature is used to preserve the
living cells and tissue.
• In this technique, tissues can be preserved for a very long time.
• The science which deals with cryopreservation is known as
“cryobiology”.

4. Principle
• The cryopreservation principle is to bring the plant cell and
tissue culture to a ‘0’ metabolism or non- dividing state by
reducing the temperature in presence of cryopreservation.
• Cryopreservation means the storage of a germplasm at a very
low temperature.
Solid carbon dioxide (at -79°C)
Low-temperature deep freezer (at -80°C)
In vapor phase nitrogen (at -150°C)
In liquid nitrogen (at -196°C)
Most commonly used cryopreservation is liquid Nitrogen at (-196°C). The cell stays in inactive
and thus the cryo can be conserved for long periods.

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

6. Precautions/Limitation
Good technical and theoretical knowledge of living plant cells as well
as cryopreservation is essential for successful cryopreservation.
> Formation of ice crystal inside the cell should be prevented
.
> High intracellular concentration of solutes may also
cells.
> Sometimes, certain solutes from the cell may leak out
freezing.

7. Technique
The Cryopreservation of plant cell culture followed by the regeneration of
plants broadly induces the following stages
• Development of Sterile Tissue Culture
• Addition of cryoprotectants and pre-treatments
• Freezing
• Storage
• Thawing
• Reculture
• Measurement of survival / viability
• Plant Regeneration

8. Organo genic Differentiation
Organ genic differentiation is an outcome of the process of
dedifferentiation followed by re-differentiation of cells.
Within the plant body, this programme is realised by integrating the network
of relations between apical and axillary shoot and root meristems, different
organs, tissues and cells.
Organogenesis -
The process of morphogenesis involving the
formation of plant organs. i.e., shoots , roots , flowers, buds from
explant or cultured plants.

9. Types of Organogenesis
• Direct Organogenesis – Tissue from leaves, stems, roots can be
directly cultured to produce plant organs.
For appropriate organogenesis in culture exogeneous addition
growth regulators – auxins and cytokinin is required.
• Indirect Organogenesis - When it occurs ,through the callus or
suspension cell culture formation.
The culture may be grown in liquid or solid medium (MS, White
can be used in organogenesis)

10. . Direct Organogenesis Indirect Organogenesis
Formation of organs directly to produce plant organs. Inverting through callus stage.
Explant – Meristemoid -Primordium Explant –Callus –Meristemoid –Primordium
Eg . Peanut Eg . Assparagus , Coffee leaf
No Callus formation Callus formation

11. Direct Organogenesis Indirect Organogenesis

12. Stages
• Dedifferentiation results in the formation of callus
from the explant tissue with accelerated cell
division.
• Whereas, redifferentiation causes the
development of primordia from a group of callus
cells.

13. References
• Biotechnology Book – U.Satynaryan
• Google