**4. The use of proline as a cryoprotectant**

Proline has been used for many years in numerous cryoprotection protocals for the preservation of a wide range of both animal and plant cells and tissues. For example, Li et. al. (2003) investigated the effects of addition of proline, glutamine, and glycine to the Tes-Tris-egg yolk (TTE) freezing medium used for cryopreservation of cynomolgus monkey (*Macaca fascicularis*) spermatozoa. They found that the addition of 5 mM proline, 10 mM glutamine, and 10 or 20 mM glycine to TTE significantly improved post-thaw sperm motility and membrane integrity compared to controls without an amino acid. Of the three amino acids tested proline was effective at the lowest concentration.

Proline has also been found to be useful for the cryopreservation of plant cells, meristems and embryos. Jain et al. (1996) included proline in the cryoprotectant solution as part of a protocol that was used to successfully cryopreserve embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties using a simple one-step freezing procedure that did not require a controlled-rate freezer. Brison et al. (1995 ) used a preculture medium enriched with dimethylsulfoxide and proline prior to the cropreservation of *in vitro* grown interspecific Prunus rootstock, Fereley-Jaspi (R). In a study to develop a cryoprotection protocol for highly

Proline and the Cryopreservation of

**5.2 Proline as an osmolite** 

references therein).

**5.3 Proline as precursor for other molecules** 

**5. Proline a multifunctional cryoprotectant?** 

**5.1 Possible mechanisms of protection** 

Plant Tissues: Functions and Practical Applications 421

and 64-100% for males. The morphology of the sporophytes formed from the cryopreserved gametophytes appeared normal and the authors suggested that this cryopreservation

Because of its ability to act as an osmoprotectant without interfering with normal cellular processes and biochemical reactions proline has been used in a range of different cryopreservation protocols both for animal and plants cells and tissues, however the exact mode by which protection is achieved is still a matter of considerable debate in the scientific literature. Proline could potentially acting as storage reserve of carbon and nitrogen, a compatible osmolyte, a buffer for cytosolic pH, a scavenger of reactive oxygen species (ROS) and as an aid to balancing cellular redox status (Smirnoff & Cumbes 1989; Hare & Cress, 1997). It has also been proposed that proline could act as a molecular chaperone, helping to stabilize the structure of proteins, and as part of the signal transduction chain alerting plant cells to the

The osmoregulatory role of proline in plant cells exposed to hyperosmotic stress has been the subject of numerous studies and under environmental conditions that result in cellular dehydration such as drought, freezing or extreme salinity, it is widely accepted that proline accumulates and acts as a compatible solute helping to protect cells from damage (Heur, 1994). Accumulation of cytoplasmic osmolytes, such as proline, is thought to aid in reducing the cellular water potential to a level below the external water potential, this enables water to move into the cell and be maintained there, while at the same time minimising potentially deleteriously increases in ionic strength. However, there is some debate in the published literature as to whether increased cytosolic levels of free proline has any direct adaptive value (Heur, 1994). While there are many reports of positive correlations between the capacity for proline accumulation and dehydration and cold tolerance (see section 3.1), some researchers still challenge the value of the ability of plant cells to accumulate proline as a positive index for osmotic stress resistance (Heur, 1994 &

It has been suggested that stress-induced accumulation of amino acids like proline may not only have an osmoregulatory role, but that they could also be a mechanism to provide cells with a pool of the precursors required to synthesis other molecules known to be involved in biotic and abiotic stress responses (Sanchez et al., 2008). For example polyamines can be synthesized from arginine or ornithine and ornithine from glutamate, hence the pathways for proline and polyamine biosynthesis are interlinked, and both groups of molecules are important in plant stress responses (Groppa & Benavides 2008). Little is known about the roles of polyamine metabolism in the process of cryopreservation, but Ramon et al. (2002) reported that an increase in putrescine content was positively correlated with the survival

rate after simple freezing or after vitrification of banana meristem cultures.

method could be used to preserve culture stocks of *U. pinnatifida* for mariculture.

presence of a stressor and hence triggering adaptive responses (Maggio et al. 2002).

freezing sensitive *Begonia* species, Burritt (2008) found that adventitious shoots of the rhizomatous begonia, *Begonia x erythrophylla* were sensitive to dehydration and very sensitive to freezing. While pre-treatment with 0.75 M sucrose significantly increased the percentage of encapsulated shoots surviving dehydration, pre-treatment with sucrose did not afford cryoprotection without prior dehydration.

Fig. 3. The percentage of *Begonia x erythrophylla* shoots surviving pre-treatment with ABA (3.8 uM) and/or proline (2.15 mM). The different letters indicate statistically different values at p < 0.05 (modified from Burritt 2008).

Addition of abscisic acid (ABA) and proline to the pre-treatment medium significantly improved the percentage of shoots surviving freezing. Pre-treatment of shoots with a medium containing, 0.75M sucrose, 3.8 μM ABA and 2.15 mM proline resulted in greater than 50% of shoots surviving freezing (Figure 3).

Christianson (1998) used a 3-4 day preconditioning treatment using a tissue culture medium supplemented with 10-5 M ABA and 100 mM proline to greatly increase survival rates and simplify a protocol for moss cryopreservation. Pretreatment with the combination of proline and ABA was used as part of a cryopreservation protocol that could be used for *Ceratodon purpureus*, *Funaria hygrometrica*, *Physcomitrella patens*, and two species of *Sphagnum*. Cryopreserved cultures remained viable at least one year at -80oC.

In addition to both animal and plant cells, proline has been found to be particularily useful for the cryopreservation of algal cells. Kuwano et al. (2004) found the gametophytic cells of six species of Laminariales, *Laminaria japonica* Areschoug, *L. longissima* Miyabe, *Kjellmaniella crassifolia* Miyabe, *Ecklonia stolonifera* Okamura, *E. kurome* Okamura, and *Undaria pinnatifida* (Harvey) Suringar could be cropreserved using a cryoprotective solution containing ethylene glycol and proline. The cells were suspended in a mixture of ethylene glycol and proline, and slowly cooled to -40oC over a period of 4 h. After a cooling step, the cells were immediately immersed in liquid nitrogen. Viabilities ranged from 36.2% to 67.2%. Nanb et al. (2009) developed a cryopreservation protocol for gametophyte strains of the edible macroalgae *Undaria pinnatifida* (Harvey). Following a pretreatment involving exposure of male and female gametophytes to low levels of light, they used a two-step cooling method with a mixture of cryoprotectants including 10% L-proline and 10% glycerol, before freezing in liquid nitrogen. Gametophyte survival rates were high, ranging from 43-60% for females

freezing sensitive *Begonia* species, Burritt (2008) found that adventitious shoots of the rhizomatous begonia, *Begonia x erythrophylla* were sensitive to dehydration and very sensitive to freezing. While pre-treatment with 0.75 M sucrose significantly increased the percentage of encapsulated shoots surviving dehydration, pre-treatment with sucrose did not afford

Fig. 3. The percentage of *Begonia x erythrophylla* shoots surviving pre-treatment with ABA (3.8 uM) and/or proline (2.15 mM). The different letters indicate statistically different values

Addition of abscisic acid (ABA) and proline to the pre-treatment medium significantly improved the percentage of shoots surviving freezing. Pre-treatment of shoots with a medium containing, 0.75M sucrose, 3.8 μM ABA and 2.15 mM proline resulted in greater

Christianson (1998) used a 3-4 day preconditioning treatment using a tissue culture medium supplemented with 10-5 M ABA and 100 mM proline to greatly increase survival rates and simplify a protocol for moss cryopreservation. Pretreatment with the combination of proline and ABA was used as part of a cryopreservation protocol that could be used for *Ceratodon purpureus*, *Funaria hygrometrica*, *Physcomitrella patens*, and two species of *Sphagnum*.

In addition to both animal and plant cells, proline has been found to be particularily useful for the cryopreservation of algal cells. Kuwano et al. (2004) found the gametophytic cells of six species of Laminariales, *Laminaria japonica* Areschoug, *L. longissima* Miyabe, *Kjellmaniella crassifolia* Miyabe, *Ecklonia stolonifera* Okamura, *E. kurome* Okamura, and *Undaria pinnatifida* (Harvey) Suringar could be cropreserved using a cryoprotective solution containing ethylene glycol and proline. The cells were suspended in a mixture of ethylene glycol and proline, and slowly cooled to -40oC over a period of 4 h. After a cooling step, the cells were immediately immersed in liquid nitrogen. Viabilities ranged from 36.2% to 67.2%. Nanb et al. (2009) developed a cryopreservation protocol for gametophyte strains of the edible macroalgae *Undaria pinnatifida* (Harvey). Following a pretreatment involving exposure of male and female gametophytes to low levels of light, they used a two-step cooling method with a mixture of cryoprotectants including 10% L-proline and 10% glycerol, before freezing in liquid nitrogen. Gametophyte survival rates were high, ranging from 43-60% for females

cryoprotection without prior dehydration.

at p < 0.05 (modified from Burritt 2008).

than 50% of shoots surviving freezing (Figure 3).

Cryopreserved cultures remained viable at least one year at -80oC.

and 64-100% for males. The morphology of the sporophytes formed from the cryopreserved gametophytes appeared normal and the authors suggested that this cryopreservation method could be used to preserve culture stocks of *U. pinnatifida* for mariculture.
