**7. References**

408 Current Frontiers in Cryobiology

due, as previously proposed by Perez-Pe et al., (2002), to a membrane modifications that determine conformational changes of these proteins or facilitate calcium influx into the cell (Bailey & Buhr, 1994; McLaughlin & Ford, 1994). This ion could stimulate adenylyl cyclase to initiate cAMP-mediated phosphorylation of sperm protein. Alternatively (or in addition), the cryopreservation procedure could also determine the activation of protein kinases

Fig. 9. Motility-dependent phosphorylation/dephosphorylation at tyrosine residues (A), threonine residues (B), and serine residues (C) in frozen–thawed sperm of gilthead sea bream before and after motility activation. Sperm were either activated in seawater (lane 1) or maintained immotile by dilution in non-activating medium (lane 2). Sperm proteins were subjected to Western blotting (30 g/lane) with antibody. Number on the left indicates molecular mass of bands. On the right, the names of proteins of interest are indicated.

Cryopreservation, coupled with insemination and short term storage techniques, will lead to an improvement of gamete management of marine fish species. In particular, sperm cryopreservation is considered as a valuable technique for artificial reproduction and genetic improvement since it allows the selection and the storage of gametes of high quality. However, although seawater fish spermatozoa of marine fish are more resistant than freshwater species to the dynamic changes in osmotic pressure that occur during the process of cryopreservation (Dzuba & Kopeika, 2002), the freezing-thawing procedure, apart from the experimental protocol used and from the fish species considered, determines: a changes of the kinematic characteristics, damages to proteins and DNA, lipid modification and change of the phosphorylation state of proteins involved in sperm motility initiation. The knowledge of the effects of freezing–thawing procedure on spermatozoa is very important to improve cryopreservation techniques for semen of marine fish for the establishment of sperm cryobanks that could play a crucial role in the genetic management and conservation

different from PKA (Pommer et al., 2003).

(Modified from Zilli et al 2008; Criobiology)

**6. Conclusions** 

of aquatic resources.


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**Part 7** 

**Cryopreservation of Plants** 


**Part 7** 

**Cryopreservation of Plants** 

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**14** 

*Australia* 

**Current Issues in Plant Cryopreservation** 

Pui Ye Phang1,2, Arwa Al-Hanbali1,2, Eric Bunn2,3 and Ricardo L. Mancera1 *1Curtin Health Innovation Research Institute, Western Australian Biomedical Research* 

Plant cryopreservation involves the storage of plant tissues (usually seed or shoot tips) in liquid nitrogen (LN) at -196°C or in the vapour phase of LN at -135°C in such a way that the viability of stored tissues is retained following re-warming (Day et al., 2008; Hamilton et al., 2009). Cryopreservation is usually applied to species with recalcitrant (i.e. dehydration sensitive) seeds that are not storable by any other means, or preservation of specific cultivars of vegetatively propagated crop plants like banana or potato, or for unique ornamental genotypes (Halmagyi et al., 2004; Kaczmarczyk et al., 2011a; Panis et al., 2005). Another reason to utilise cryostorage is to conserve endangered plant species, particularly where seeds may be extremely scarce or of doubtful quality and/or the species is threatened with imminent extinction (Decruse et al., 1999; Mallon et al., 2008; Mandal & Dixit-Sharma, 2007;

The main advantage of cryopreservation is that once material has been successfully cooled to LN temperatures, it can be conserved in principle indefinitely, because at these ultra-low temperatures no metabolic processes occur. Replenishing a small volume of LN weekly in cryo-dewars is the only on-going maintenance operation usually required in cryostorage. There are further advantages to this approach: the low costs of storage, minimal space requirements and reduced labour maintenance compared to living collections and even when compared to maintenance of tissue cultures at room temperature. Once in storage, there is no risk of new contamination by fungus or bacteria, and cryogenically stored material has been reported to retain genetic stability (Harding, 2004). Depending on the species, small cryopreserved samples may take several weeks to re-establish shoot cultures, and several months to a year may be required to produce micropropagated plants capable of transfer to

Shoot tips (containing the apical meristem) are the most commonly used plant material for cryostorage. The apical meristem is composed of small unvacuolated cells served with a relatively small vascular system. The organised structure of apical meristems generally results in direct shoot formation after re-warming, thereby maintaining the genetic integrity

Paunescu, 2009; Sen-Rong & Ming-Hua, 2009; Touchell et al., 2002).

soil under greenhouse conditions and (following weaning) into the field.

**1. Introduction** 

Anja Kaczmarczyk1,2, Bryn Funnekotter1,2, Akshay Menon1,2,

*2Botanic Gardens and Parks Authority, Fraser Avenue, West Perth 3School of Plant Biology, Faculty of Natural and Agricultural Sciences,* 

*Institute, Curtin University, Perth* 

*University of Western Australia* 
