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According to Villalobos and Engelmann (1995), over 1200 institu-
tions worldwide have established germplasm collections in field gene
banks and botanical gardens. These institution include the Consulta-
tive Group on International Agriculture Research Centers (CGIAR) (Vil-
lalobos and Engelmann 1995), the International Board for Plant Genetic
Resources (IBPGR) (Withers 1991), the Botanic Garden of Siena (Sgarbi
et al. 2001), and the Tropical Gardens and Research Institute, India
(Sarasan et al. 2006).
However, limitations ofex situconservation include large space and
specialized facilities needed, considerable labor, and limited number of
species. Field gene banks are also subjected to environmental stresses,
climate change, and various plant diseases (Pennycooke and Towill
2000; Matsumoto et al. 2001).
Biotechnology offers two alternative techniques to conserve plant
species;in vitroculture and cryopreservation.In vitroconservation
approaches require small space in culture rooms for maintaining thou-
sands of genotypes without the threat of diseases and pest attacks (Uyoh
et al. 2003). However, they are limited to short- and medium-term stor-
age. Genotypes could possibly be lost due to contamination or human
error during the process (Engelmann 1991; Escobar et al. 1998; Charoen-
sub et al. 1999). In addition, genetic instability might become a problem
when the plants are maintainedin vitroover some period of time (Rao
2004).
Cryopreservation is a viable long-term alternative and the technique
involves the storage of live plant cells, tissues, or organs at ultra-low
temperatures (–196◦C in liquid nitrogen or –150◦C in nitrogen’s vapor
phase) with a supposed low risk of genetic and physiological changes
even over long periods of time (Gonzalez-Benito and Perez 1997; Reed
2008). Cryopreservation is not a substitute for field maintenance but it
provides a suitable backup against the loss of valuable genotypes that
may occur under field conditions. It is reliable for maintaining viabil-
ity of germplasm under stable and low cost conditions, and safe from
diseases or environmental damage (Harvengt et al. 2004). Shoot tips,
somatic and zygotic embryos, whole seeds, pollen, anther, and buds
can be cryopreserved and stored.
Classical cryopreservation techniques, which are based on freeze-
induced dehydration, are mainly employed for freezing undifferenti-
ated cultures and apices of cold-tolerant species (Engelmann 2004).
This involves slow cooling to a defined prefreezing temperature,
followed by rapid immersion in liquid nitrogen. With temperature
reduction during slow cooling, cells and the external medium initially
supercool, followed by ice formation in the medium (Mazur 1984).