CryoLetters 26 (2), 121-130 (2005) © CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

SURVIVAL OF FOUR ACCESSIONS OF manglesii () SEEDS FOLLOWING EXPOSURE TO LIQUID NITROGEN

D. J. Merritt1*, D.H. Touchell2, T. Senaratna1,3, K.W. Dixon1,3 and C. W. Walters4

1Kings Park and Botanic Garden, West , WA 6005, Australia. 2School of Forestry and Wood Products, Michigan Technological University, Houghton, MI 49931-1295, USA. 3Faculty of Natural and Agricultural Sciences, The University of , Crawley, WA, 6009, Australia. 4USDA-ARS National Centre for Genetic Resources Preservation, 1111 South Mason St, Fort Collins, CO 80521, USA. *Correspondence: [email protected]

Abstract This study investigated the survival of seeds from the prominent endemic Western Australian Anigozanthos manglesii following exposure to liquid nitrogen (cryostorage). Seeds from four different accessions (collected in 1987, 1990, 1993 and 1998) adjusted to different water contents were tested for survival following cryostorage. Water content was a significant determining factor with survival of cryostored seeds declining rapidly at water contents above c. 18%. These water contents were deemed as critical water contents and were supported by DSC scans showing high endothermic peaks indicating ice crystallisation. In some instances, survival of cryostored seeds also declined at low water contents. Seeds from 1990 had a lower than expected survival compared to the other accessions. This may have resulted from the higher lipid content of seeds from this accession, or the reduced germination and vigour of these seeds prior to cryostorage. Keywords: Anigozanthos manglesii, conservation, cryostorage, seed, water content.

INTRODUCTION Seed banks for conservation purposes must ensure the long-term survival of an accession. Seeds of wild species may be in short supply and, for threatened species in particular, there may be limited collection opportunities and few seeds available to form a collection. Therefore, for valuable germplasm it is essential to provide the best possible storage conditions as any loss of seed viability during storage may impact significantly on the number of available for restoration projects in future years. Cryostorage offers an ideal method for germplasm conservation as there is the potential for extended storage periods (possibly many hundreds or thousand of years) with no loss of viability (13,27). The development of methods for cryostorage of seeds initially centred on species of agricultural importance (10,12,20). More recent studies have demonstrated the potential of cryostorage for conservation of wild species (4,7-9,17,18) and cryostorage is viewed as an important tool for the conservation of priority species in Australia (19). Seed survival following cryostorage is highly dependent upon seed water content. Many studies have documented the existence of a critical water content (sometimes termed the high moisture freezing limit) below which successful cryostorage is not constrained by cooling rates or addition of cryoprotectants. Exposing seeds to liquid nitrogen above the critical water content results in lethal ice crystal formation (2,10,14,21). Some species also show damage at water contents much lower than the critical threshold associated with ice-crystal formation 121 (7,13,20,27). Therefore, resolving the optimal water content(s) is essential for successful cryostorage. The effects of seed age on survival of cryostorage have received less attention, although a previous study on Banksia ashbyi demonstrated that freshly collected seeds were more able to survive cryostorage at water contents exceeding the critical threshold, compared to older seeds (7). Many seed banks have seed accessions that have been stored under conventional conditions for a number of years. For greater security and longevity of important germplasm (e.g. that of rare and endangered species) it may be beneficial to transfer these collections to cryostorage to maximise longevity, provided the seed quality is not adversely affected by such treatment. This study investigates cryostorage of seeds of Anigozanthos manglesii D. Don (Haemodoraceae), a species representative of an important endemic Western Australian comprising 25 species, nine of which are currently stored in the seed bank at Kings Park and Botanic Garden, Western Australia, with accessions dating back to 1972. Specifically, this study examines: the ability of seeds from different accessions to survive cryostorage; whether the critical water content, above which survival is significantly reduced, varies between seeds from different accessions; whether variations in survivability and the critical water content are related to seed lipid content; and, for seeds of one accession, thermal transitions and electrolyte leakage in relation to survival following cryostorage at different water contents.

MATERIALS AND METHODS Seed material Seeds of Anigozanthos manglesii from four different accessions were obtained from the seed store at Kings Park and Botanic Garden, in Perth, Western Australia. Seeds collected in 1987, 1990, 1993 and 1998 were selected. Prior to the experiments in this study (conducted in 1998 and 1999), seeds collected in 1987 and 1990 were stored air-dry at c. 23°C and seeds collected in 1993 were stored at -14°C. Seeds collected in 1998 were used immediately. All seeds were collected from within bushland at Kings Park and Botanic Garden.

Seed germination testing Freshly collected Anigozanthos manglesii seeds have an after-ripening requirement for germination to proceed. This can be circumvented by heat-pulsing seeds at 100°C for 3 h (16). Prior to any experimentation, all seeds were heat-pulsed to remove the confounding factor of the influence of seed dormancy on germination of seeds of different ages. Following the heat-pulse, seed water content was adjusted by placing seeds over saturated solutions of ZnCl2 (5% RH), LiCl (13% RH), MgCl2 (32% RH), K2CO3 (43% RH), Ca(NO3)2 (50% RH), NaCl (75% RH), KCl (85% RH) and KNO3 (92% RH) (24, 29) at 23°C for 4 weeks. For the highest water contents tested (> 20%) seeds were placed over de-ionised water for varying -1 periods between 7 - 14 h. Seed water content was determined gravimetrically (g H2O g DW) by drying three replicates of 50 seeds in an oven at 103°C for 17 h (6). Following adjustment of seed water content, seeds were removed for immediate germination testing (non- cryostored), or placed in 2 ml polyethylene cryovials (Nunc®), plunged directly into liquid nitrogen and stored for 1 week, then warmed at room temperature for 20 min before further handling. To test for germination, seeds were sterilised in a 2% calcium hypochlorite solution (Ca(OCl)2) for 15 mins, washed in sterile de-ionised water, and incubated on sterile water agar plates (0.7% w/v) in the dark at 18°C. Five replicates of 10 seeds were incubated for each treatment. Seeds were considered germinated upon radicle emergence and germination was scored every 3 d, from the time the first seeds germinated, for 3 weeks, at which time maximum germination had occurred. Total seedling length was measured after 3 weeks. Time to initial germination (t0) and time to 50% maximum germination (t50) were determined from cumulative germination curves derived from the mean values of the five replicates.

122 Seed lipid content Seed lipid content was determined following the methods of Senaratna et al. (11). Three replicates of 50 seeds (at air dry water contents) were homogenised in chloroform:methanol (2:1 by volume) and centrifuged at 12 000 rpm for 15 min. The supernatant was partitioned with 0.7% (w/v) NaCl and centrifuged again at 10 000 rpm for 10 min. The organic layer was -1 evaporated under N2 and residual lipids determined gravimetrically (g lipid g FW).

Differential scanning calorimetry For seeds collected in 1993, warming thermograms of seeds at different water contents were measured via DSC analysis, using a Perkin Elmer DSC7. Between 5 - 8 seeds (giving a fresh weight of c. 30 mg) were used for each test. Following adjustment of seed water content, seeds were sealed in aluminium pans and cooled at 10°C min-1 from 20°C to -150°C, then warmed at the same rate (25). Thermal transitions were measured as endothermic (melting) peaks in the baseline and the enthalpy of the transitions calculated as the area under the peak. The onset temperature of a transition was determined by the intersection of the baseline and the tangent to the steepest part of the peak. All calculations were made using Perkin Elmer software.

Electrolyte leakage Electrolyte leakage was also measured for seeds collected in 1993. Seeds were heat- pulsed and equilibrated to a range of water contents as described above. Seeds were then cryostored or non-cryostored as for the germination trials and placed in vials containing 20 ml de-ionised water. The conductivity of this solution was measured over a 3 h period using a TPS 900-C conductivity meter. Three replicates of 30 seeds were used for each test.

Statistical analysis Germination, seedling length and electrolyte leakage data were analysed by analysis of variance. Germination percentage values were arcsine-transformed prior to statistical analyses (untransformed data appears in all figures). Fisher’s least significant difference (P<0.05) was used to determine significant differences between treatments.

RESULTS Seed germination Following adjustment at different relative humidities, seed water content ranged from c. 2 - 26% (Fig.1, Table 1). Germination of non-cryostored seeds collected in 1987, 1993 and 1998 averaged c. 90% (across all water contents). Non-cryostored seeds collected in 1990 had lower germination, averaging 74% (Fig.1a-d). Seed water content did not greatly affect germination of non-cryostored seeds and germination generally varied by ≤10% across all water contents, although in some instances, particularly for seeds collected in 1987 and 1990, a significant increase (P<0.05) in germination was noted at high water contents (17 – 21%), compared to lower water contents. Germination following cryostorage was highly dependent upon seed water content. The critical water content (defined as the highest water content at which seeds survived cryostorage without a significant reduction (p<0.05) in germination, compared to non-cryostored seeds) was 18% for seeds collected in 1987 and 1990, 21% for seeds collected in 1998, and 22% for seeds collected in 1993. In most cases germination was not affected by cryostorage at water contents below the critical threshold, although in some cases a small, but significant (p<0.05), reduction in germination was noted. This occurred in cryostored seeds collected in 1987 at 3.2% water content, seeds collected in 1993 at 3.5% water content, seeds collected in 1990 at 2.6% and 6.7% water content, and seeds collected in 1998 at 8.3% water content.

123 a e 100 8 m) ) c

% 75 6

50 4 ination ( m 25 2 Ger Seedling Length ( 0 0 0 5 10 15 20 25 0 5 10 15 20 25

b f 100 8 m) ) c

% 75 6

50 4 ination ( m 25 2 Ger Seedling Length ( 0 0 0 5 10 15 20 25 0 5 10 15 20 25 c g 100 8 m) ) c

% 75 6

50 4 ination ( m 25 2 Ger Seedling Length ( 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30

d h 100 8 m) ) c

% 75 6

50 4 ination ( m 25 2 Ger Seedling Length ( 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Water Content (%) Water Content (%)

Non-cryostored Cryostored

Figure 1. Germination (a-d) and seedling length (e-h) of non-cryostored and cryostored Anigozanthos manglesii seeds at different water contents. Seeds were collected in 1987 (a,e); 1990 (b,f); 1993 (c,g); 1998 (d,h). Data points represent mean ± S.E.

The rate of germination and total seedling length (after 3 weeks incubation) were also measured to provide additional estimates of seed performance. For non-cryostored seeds of most accessions t0 and t50 generally occurred at the same time (9 days after incubation), regardless of seed water content, although for seeds collected in 1990 t50 was reached 1 - 2 days after t0 in most cases (Table 1). Cryostorage at water contents up to the critical threshold 124

Table 1. Rate of germination (time for first seed to germinate (t0) and time for 50% of seeds to germinate (t50)) of non-cryostored and cryostored Anigozanthos manglesii seeds at different water contents.

Non-cryostored Cryostored

Year Water Content (%) t0 (days) t50 (days) t0 (days) t50 (days) 1987 3.2 ± 0.0 9 9 9 9 5.4 ± 0.1 9 9 9 9 5.8 ± 0.1 9 9 9 10 7.8 ± 0.4 9 9 9 10 11.1 ± 0.6 9 9 9 9 13.0 ± 0.6 9 10 9 11 18.0 ± 1.5 9 9 9 10 19.0 ± 0.1 9 9 10 13 22.2 ± 0.3 9 9 - - 24.7 ± 1.1 9 9 - - 1990 1.7 ± 0.2 9 11 9 12 2.6 ± 0.2 9 10 9 12 6.7 ± 0.5 9 10 9 12 8.2 ± 0.4 9 9 9 11 9.3 ± 0.4 9 10 9 12 12.2 ± 0.2 9 13 12 15 17.3 ± 0.1 9 11 11 14 18.0 ± 0.3 9 11 14 15 21.3 ± 0.7 9 9 - - 21.5 ± 0.3 9 9 18 18 1993 3.5 ± 0.1 9 9 9 11.5 5.7 ± 0.1 9 9 9 9 5.7 ± 0.6 9 9 9 10 7.4 ± 0.4 9 10 9 10 8.2 ± 0.5 9 9 9 9 12.0 ± 0.2 9 9 9 10 15.7 ± 1.0 9 10 9 11 17.1 ± 0.8 8 8 9 9 19.7 ± 0.9 9 10.5 9 11.5 22.1 ± 0.7 9 9 10 10 22.2 ± 1.2 8 8 - - 23.8 ± 0.4 9 9 12 15 24.5 ± 0.7 9 9 - - 26.1 ± 0.9 9 9 - - 1998 3.6 ± 0.3 9 9 9 9 5.3 ± 0.7 9 9 9 10 5.5 ± 1.1 9 10 9 9 8.2 ± 0.3 9 9.5 9 11 10.4 ± 1.9 9 9 9 9 12.9 ± 0.2 9 12 9 11 19.4 ± 0.4 9 9 9 9 21.2 ± 0.3 9 9 11 13 23.2 ± 0.6 10 10 - - 23.3 ± 0.6 9 9 - -

125 did not influence t0 for seeds collected in 1987, 1993 and 1998, but t50 was often delayed by 1 - 2 days. For seeds collected in 1990, cryostorage resulted in an increase in t0 at and above 12% water content (well below the critical threshold), and t50 increased to 12 – 18 days at all water contents. Total seedling length displayed a similar pattern to germination (Fig. 1e-h). For non- cryostored seeds total seedling length was similar at most water contents, although some increase was noted in seeds at water contents greater than 20%, especially those collected in 1987 and 1990. Following cryostorage at or above the critical water content a significant decrease (compared to non-cryostored seeds) in seedling length was noted. Below the critical water content seedling length remained similar to that of non-cryostored seeds.

Seed lipid content The lipid content of seeds collected in 1990 was 22 ± 2%, considerably higher than that of seeds collected 1987, 1993 and 1998 (16 ± 2%, 12 ± 2% and 16 ± 3%, respectively).

Differential scanning calorimetry Using DSC, warming thermograms of seeds collected in 1993 were obtained over a water content range of 2 - 40%. Representative thermograms are presented in Figure 2.

5

4 3% ) 3 r (mW

e 10% 2 Pow

1 20%

0 5

4 28% ) 3 r (mW e 2 Pow

1 Endothermic

0 15

37%

) 10 r (mW e

Pow 5

0 -160 -140 -120 -100 -80 -60 -40 -20 0 20 Temperature (°C)

Figure 2. Representative warming thermograms of Anigozanthos manglesii seeds (collected in 1993) at indicated water contents. Samples were cooled at 10°C min-1 to -150°C and then warmed at 10°C min-1. Note the difference in scale on the y-axes.

At water contents below 20%, endothermic peaks with an energy of approximately 10 J g-1 DW and an onset temperature of -16°C were measured. Immediately preceding these peaks,

126 in many scans a small exothermic peak, with an energy of 0.5 – 1.0 J g-1 DW was evident. Above 20% water content, endothermic peaks were measured which increased considerably in size and sharpness when water content increased. The onset temperature of these peaks remained relatively constant, between -16°C to -17°C. A plot of the energy of the melting events versus seed water content shows the constant energy of the endothermic peaks at water contents up to 20%, followed by a linear increase (r2 = 0.90) in the energy of the peaks between 20 - 40% water content (Fig. 3).

60

50

40 DW) -1 30

20 Energy (J g

10

0 0 0.1 0.2 0.3 0.4 -1 Water Content (g H2O g DW )

Figure 3. Effect of water content on the enthalpy of the endothermic transition in Anigozanthos manglesii seeds (collected in 1993). Transition enthalpies were calculated by determining the area under the peak from warming thermograms similar to those presented in Fig. 2.

Electrolyte leakage The rate of electrolyte leakage from seeds collected in 1993 was determined following cryostorage at a range of water contents above and below the critical threshold. Conductivity of the imbibing solution increased approximately linearly (r2 = 0.81 - 0.98) between 5 and 180 mins. An example for seeds of 25% water content is presented in Figure 4. There were no significant differences (P<0.05) observed between the rate of leakage of cryostored and non- cryostored seeds, regardless of seed water content (Table 2).

Table 2. Rate of electrolyte leakage (mean ± S.E.) of Anigozanthos manglesii seeds (collected in 1993) non-cryostored or cryostored at different water contents.

Water Content (%) Rate of Leakage (µS cm-1 h-1 g dw-1) Non-cryostored Cryostored 5.5 ± 1.3 23.3 ± 3.2 30.5 ± 5.1 11.1 ± 1.6 25.1 ± 4.0 23.8 ± 3.5 20.8 ± 0.3 27.4 ± 4.1 27.6 ± 1.8 25.0 ± 1.5 27.5 ± 6.4 23.6 ± 0.7 29.5 ± 1.3 23.3 ± 2.3 24.8 ± 1.7

127 250

200 DW) -1

g 150

-1 Non-cryostored

S cm Cryostored 100

50 Leakage (µ

0 0 30 60 90 120 150 180 Time (mins)

Figure 4. Electrolyte leakage from non-cryostored and cryostored seeds of Anigozanthos manglesii (collected in 1993) at 25% water content. Data points represent mean ± S.E.

DISCUSSION The results of this study demonstrated that Anigozanthos manglesii seeds collected in 1987, 1993 and 1998 generally survived exposure to liquid nitrogen with no evidence of immediate detrimental effects on germination or seedling vigour, provided seed water content was below a critical threshold of 18–22%. These values are similar to that of many other species tested (2,7,14). Seeds cryostored above this critical water content showed a pronounced decrease in germination, with a concurrent decrease in the germination rate and seedling length, indicating that seedlings from those seeds which did survive had reduced vigour. A small, but significant reduction in germination was also observed in some seeds cryostored at low water contents (c. 3–6%). Reduced germination following cryostorage at low water contents has previously been noted in rapidly cooled, lipid-rich seeds of sesame (13), soybean and sunflower (20) seeds. Although in this study a consistent trend of decreased germination of cryostored seeds at very low water contents was not demonstrated, it is apparent the combined effects of desiccation and cryostorage may sometimes damage seeds, and excessive drying prior to cryostorage should be avoided. Seeds collected in 1990 were of poorer quality than those collected in other years, with a lower germination percentage prior to cryostorage (c. 15% less than other accessions) and a lower total seedling length (c. 7-15% less). The reduced initial quality of these seeds appeared to impact on the germination following cryostorage, as the germination rate of cryostored seeds was lower at all water contents. Therefore older seeds, and/or those of poor initial quality, may have a reduced tolerance to the stresses imposed by cryostorage. A previous study investigating cryostorage of Banksia ashbyi seeds of various ages demonstrated that seed germination above the critical water content was greatest in freshly collected seeds (7). In the present study, the fact that germination performance of cryostored seeds collected in 1987 was greater than that of seeds collected in 1990 suggests that initial seed quality (viability), rather than seed age per se, may be the more important factor. Variations in seed composition between different accessions may also influence seed survival of cryostorage. For example, several studies demonstrate that lipid reserves in seeds may interact with water and influence survival (20,21,30). A recent study demonstrated a negative correlation between unfreezable water content and lipid content for seven Coffea species (2). Such a clear correlation was not demonstrated in the present study, but there was perhaps some evidence of an influence of seed lipid content on the critical water content, as seeds collected in 1990 had 128 the highest lipid content and the lowest critical water content. At a given water content seeds with a higher lipid content have a higher water activity and, thus, increased water mobility (24). This contributes to ice formation and reduced survival following cryostorage at lower water contents. The DSC data gathered on the thermal properties of A. manglesii seeds collected in 1993 during warming from -150°C correlates well to the germination data of this accession. Between water contents of 2–19%, endothermic peaks of constant energy (10.0 J g-1 DW) are visible. These peaks are attributed to lipid (triacylglycerol) peaks as the melt energy remains constant as water content increases (20,22). The seeds contain approximately 12% lipid, thus the energy of the melt (10.0 J g-1 DW) equates to an energy of approximately 83 J g-1 lipid, similar to that found for lipids extracted from seeds of lettuce and tomato (67 and 78 J g-1 lipid, respectively) (23). The rapid increase in energy of the endothermic peaks with increasing water content above 22% is attributed to the melting of frozen water (21,22). A sharp peak is also present in some scans at around 0°C. Similar peaks have been related to lethal freezing events in Camellia sinensis (28) and embryos of Taxus brevifolia seeds (26). At water contents greater than 19%, the enthalpy data was separated into two regions (22) between 19 – 26% water 2 -1 2 -1 content (r = 0.79, slope 124 J g H2O) and >26% water content (r = 0.87, slopes 259 J g H2O). The water content at which freezable water is first detected was calculated from the point of intersection of the slope between 19 – 26% water content and the energy associated with the non-water (triacylglycerols) melt. This water content is 18%, providing a good correlation between detectable water freezing events and the critical water content determined via germination testing. Therefore, it appears A. manglesii seeds are unable to survive any intracellular ice formation. Notably, although there was an increase in the energy of water melts in seeds frozen at lethal water contents, the rate of electrolyte leakage from seeds following cryostorage was not related to seed water content or survival. The plasma membrane of cells is thought to be a primary site of freeze injury (15). Physical damage to cell membranes by ice crystals, or phase changes of the lipid components of membranes during cryostorage and re-warming are thought to disrupt membrane functioning and result in increased solute leakage from cells (1, 3, 15). The lack of an increase in solute leakage in the present study suggests that freeze- induced injury in cells of A. manglesii seeds may have occurred without severe or irreversible disruption to the plasma membrane. Alternatively, the small size of A. manglesii seeds may be a contributing factor, as leakage tests are often recommended for larger seeds (5) and the test applied in this study may not have been sensitive enough to detect subtle differences in solute leakage of small seeds. However, a similar lack of relationship between solute leakage and survival following cryostorage was noted for another Western Australian species, Banksia ashbyi (7), and the seeds of this species are much larger, suggesting electrolyte leakage methods may not be suitable for assessing freeze damage and indicating tissue viability in some seeds. Cryostorage has proved to be a potentially viable method of long-term storage for A. manglesii seeds from a number of accessions. In general, initial seed germination and seedling vigour were unaffected by cryostorage, provided seed water content was below the critical threshold. However, the results suggest that seeds with poor initial quality may have reduced tolerance to cryostorage, especially at higher water contents approaching the critical threshold. Thus there remains a need to carefully regulate the water content of seeds prior to cryostorage.

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Accepted for publication 27/3/05

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