**11. Cryopreservation**

The word is derived from two Greek words. i.e., Kryos means frosting while preservation means storage for a long time or increasing half-life in one way or another. Following these techniques cells and tissues are stored at a much lower or frozen temperature either using carbon dioxide at −79 °C or nitrogen gas at −160 °C in the form of vapors in deep freezers. In the c as e of liquid nitrogen, the limit of temperature would be from 170 °C to 197 °C. The technique involves four stages involving freezing, thawing, and re-culturing, etc. [58]. Thus, freezing temperature inactivates the cells and tissues so that it can be preserved for a longer period. Any of the tissues of the plant can be preserved under proper conditions for example meristem, stem, ovules, anther, embryos, endosperm, cells, and leaves, etc. [59].

The process of cryopreservation being followed by regeneration of the whole plant invoke the following steps **Figure 2**;


## **11.1 Isolation of sterile tissue or cell**

Physiological and structural conditions of plant effects ultimately the survival of the plant during cryopreservation. Tissues to be used in preservation must be healthy, small, young, having rich cytoplasm, and highly vacuolated. In either case, callus acts as the best source of tissues as it is more resistant to damage caused by freezing [60]. So, a callus after 1 to 2 weeks of subculturing is selected for the

#### **Figure 2.**

*Schematic flow sheet representation of steps involved in cryopreservation of plant materials in biotechnology for genetic resource(germplasm) conservation of endangered plant species such as golden paintbrush from natural environment to cryobank in form of seeds, tissues, roots, meristem and shoots etc.*

cryopreservation process. But old and black areas should be avoided and organized structures are preferred more.
