- Email: [email protected]
Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modified Vitrification Protocol
Annals of Botany 87: 371±378, 2001 doi:10.1006/anbo.2000.1346, available online at http://www.idealibrary.com on
Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modi®ed Vitri®cation Protocol S . R . T U R N E R * {{, T. S E N A R AT N A{, E . B U N N {, B . TA N {, K . W. D IX O N{ and D . H. TO U C HE L L} {Kings Park and Botanic Garden, West Perth, WA 6005, Australia, {Curtin University of Technology, Bentley, WA 6102, Australia and }USDA-ARS National Seed Storage Laboratory, 1111 South Mason St, Fort Collins, Colorado 80521, USA Received: 15 September 2000 Returned for revision: 3 November 2000 Accepted: 21 November 2000 Published electronically: 26 January 2001
An ecient vitri®cation procedure was developed and successfully applied to cryopreserve six endangered West Australian species ( family Haemodoraceae: Anigozanthos humilis ssp. chrysanthus Hopper; A. kalbarriensis Hopper; A. viridis ssp. terraspectans Hopper; Conostylis dielsia ssp. teres Hopper; C. micrantha Hopper and C. wonganensis Hopper). Species were initially evaluated for cryostorage using a basic vitri®cation protocol involving: culturing plantlets in vitro for 21 d; excision of shoot apices; preculture of apical tips on 0.4 M sorbitol for 2 d, followed by incubation in PVS2 ( plant vitri®cation solution 2) for 25 min at 0 8C, then direct immersion in liquid nitrogen (LN). Warming of retrieved material was for 1 min in a 40 8C water bath. Using this protocol ®ve of the six species exhibited low post-storage survival, while the sixth species, A. viridis ssp. terraspectans posted higher survival (61.1 %). Using A. viridis ssp. terraspectans as an indicator species, the initial protocol was modi®ed to include: 3 d preculture on 0.80 M glycerol, loading treatment with 2.0 M glycerol plus 0.4 M sucrose solution for 20 min, followed by 25 min exposure to a modi®ed PVS2. Survival was signi®cantly improved in the test species, and in further experiments three other species also showed signi®cant improvements with the new protocol. Key ®ndings include: eectiveness of glycerol in the preculture medium; the eect of preculture duration; the importance of a loading stage for these species; and the successful use of modi®ed PVS2 solutions with reduced or zero dimethyl sulfoxide # 2001 Annals of Botany Company (DMSO). Key words: A. humilis ssp. chrysanthus, A. kalbarriensis, A. viridis ssp. terraspectans, Conostylis dielsia ssp. teres, C. micrantha, C. wonganensis, kangaroo paws, Haemodoraceae, vitri®cation, cryopreservation, rare and endangered, conservation.
I N T RO D U C T I O N The Haemodoraceae (or `bloodroots') have representative taxa on several continents, including Africa, South America and North America (Hopper, 1993). In the south west of Western Australia (WA) this family shows considerable diversi®cation with 77 species belonging to seven genera. The majority of these genera i.e. Macropidia (black kangaroo paw), Anigozanthos (kangaroo paws), Blancoa, Conostylis, Phlebocarya and Tribonanthes are endemic to this region (Hopper, 1993). Amongst the endemic species of WA Haemodoraceae, many are classi®ed as rare and endangered, including seven species of Conostylis and three subspecies of Anigozanthos (Brown et al., 1998). Cryopreservation is currently being developed by agencies such as Kings Park and Botanic Garden (West Perth, Australia) to conserve threatened species. The advantages of cryostorage over conventional forms of germplasm storage include: reduction in maintenance costs associated with living collections; reduction in storage space; minimization of losses due to diseases and contamination (with living collections); and minimization of somaclonal variation (Bajaj, 1995). * For correspondence. E-mail [email protected]
0305-7364/01/030371+08 $35.00/00
For cryopreservation of endangered species, shoot apices are the preferred tissue source as they allow for the conservation of genetically stable tissues, which are essential for future re-introduction programs. Furthermore, cells found in the apical region of shoot apices are much more amenable to cryostorage protocols as they consist of small thin-walled actively dividing cells that have small vacuoles and a high nucleo-cytoplasmic ratio (Engelmann, 2000). Among several cryopreservation protocols, vitri®cation is a relatively simple method requiring no special cooling equipment (Benson, 1999). Most vitri®cation protocols utilized are based on plant vitri®cation solution 2 (PVS2) developed by Sakai et al. (1990), which involve the following steps: preculturing shoot apices on a sucrose enriched medium, loading treatment and dehydration with PVS2 prior to a plunge into liquid nitrogen (LN). Previously developed protocols for endangered Australian species involved preculturing shoot apices on a medium containing sorbitol, followed by incubation in PVS2 before direct immersion in LN (Touchell and Dixon, 1999; Touchell et al., 2001). In a previous study, Turner et al. (2000) achieved postLN survival of 36 % for Anigozanthos viridis ssp. viridis when shoot apices were precultured on a 0.4 M sorbitol # 2001 Annals of Botany Company
372
Turner et al.ÐCryopreservation of Endangered Species
medium for 2 d, before exposure to PVS2 for 25 min, followed by direct immersion in LN. This protocol was also found to be applicable to other Haemodoraceae species, although survival in all taxa tested was very low. The objectives of this study were to examine post-LN survival of Haemodoraceae species in response to: (a) alterations to preculture duration; (b) sugars/polyalcohols in the preculture medium; (c) vitri®cation solutions; and (d) the addition of a loading step. M AT E R I A L S A N D M E T H O D S
using a 0.2 mM Acrodisc1 32 ®lter prior to use. Treated shoot apices were then washed three times for 4 min per rinse, in half-strength MS liquid basal medium containing 1.0 M sucrose. After washing, shoot apices were placed onto recovery medium to assess the survival prior to LN immersion. Apices to be cooled were harvested, precultured, incubated in PVS2 as described above, plunged into LN and stored for at least 30 min prior to warming in a 40 8C water bath for 1 min. After warming, shoot apices were removed from the PVS2 medium and washed as described previously. After washing, shoot apices were placed onto recovery medium.
Species and in vitro culture The six species used in this study were: Conostylis dielsia ssp. teres, C. micrantha, C. wonganensis, Anigozanthos viridis ssp. terraspectans, A. humilis ssp. chrysanthus and A. kalbarriensis. With the exception of A. humilis ssp. chrysanthus, all species were obtained from in vitro shoot culture stocks maintained at the Plant Science Laboratory, Kings Park and Botanic Garden (Perth) and derived from ®eld collected shoots. In the case of A. humilis ssp. chrysanthus, shoot cultures were initiated from zygotic embryos. In vitro plantlets were subcultured at 3 to 4 weekly intervals on a basal medium (BM) containing half strength Murashige and Skoog (MS) salts (Murashige and Skoog, 1962) supplemented with 3.0 mM thiamine hydrochloride, 2.5 mM pyridoxine hydrochloride, 4.0 mM niacin, 0.5 mM MES buer, 0.5 mM myo-inositol, 60 mM sucrose, 0.5 mM 6benzylaminopurine (BAP) and solidi®ed with 0.8 % (w/v) agar. The pH of the medium was adjusted to 5.9 prior to the addition of agar, then autoclaved at 121 8C for 20 min. Plant cultures were incubated at 22±25 8C and illuminated with 16 h light with photosynthetic photon ¯ux density of 30 mmol m ÿ2 s ÿ1. Evaluation of the previously developed protocol In the ®rst experiment the ecacy of the protocol developed by Turner et al. (2000) was evaluated using the six study species. Turner et al.'s protocol consisted of six phases: culture, preculture, PVS2 exposure, LN immersion, warming and recovery. Shoot apices were harvested and evaluated following four key stages in the protocol: (a) immediately after harvest ( placed directly onto recovery medium without any treatment); (b) after preculture; (c) after preculture and PVS2 exposure; and (d) after preculture, PVS2 exposure, LN immersion, warming and washing. In the preculture stage, shoot apices 1.0±1.5 mm long which were excised from 19±23-d-old shoots were precultured on BM supplemented with 0.4 M sorbitol for 2 d. These shoot apices were then placed onto BM supplemented with 2 mM choline chloride recovery medium (in darkness for 7 d) (Touchell et al., 2001), with or without incubation in PVS2 (and for a sub-sample, LN immersion). Shoot apices at this stage were exposed to PVS2 at 0 8C for 25 min. PVS2 comprised 30 % w/v glycerol, 15 % w/v ethylene glycol and 15 % w/v dimethyl sulfoxide (DMSO) in half-strength MS liquid medium supplemented with 0.4 M sucrose (Sakai et al., 1990). The medium was ®lter-sterilized
Comparison of shoot apex responses ( for six dierent Haemodoraceae species) after direct exposure to PVS2 Shoot apices were harvested from all species and immediately incubated in PVS2. These apices were then rinsed in 1 M sucrose solution and placed on recovery medium. Subsequent experiments were designed to modify and improve the above protocol. Comparison of preculture duration time Shoot apices of A. viridis were precultured for 0, 1, 2, 3 and 7 d on BM containing 0.4 M sorbitol, incubated in PVS2 at 0 8C, washed and placed on recovery medium (without placing in LN) or plunged directly into LN, warmed, washed, and placed on recovery medium. Comparison of dierent sugars and polyalcohols in the preculture medium Seven dierent sugars and polyalcohols were evaluated in preculture media using Anigozanthos viridis shoot apices. In the ®rst experiment, shoot apices were precultured on BM supplemented with either 0.4 M glucose, sucrose, trehalose, ranose, glycerol, inositol or sorbitol for 3 d, then incubated in PVS2, cooled in LN, warmed and placed on recovery medium as noted above. In the second experiment, shoot apices were placed on BM supplemented with dierent molarities of these compounds. Molarities were formulated to provide the same equimolar concentrations of hydroxyl (OH) groups as 0.4 M sorbitol (14.448 1023 l ÿ1 of preculture medium). This was formulated by taking into account the total number of OH groups per molecule (e.g. sorbitol has six, glycerol has three) then adjusting the concentration to provide the same number of OH groups as 0.4 M sorbitol. Using this procedure the following concentrations were determined: 0.480 M glucose, 0.300 M sucrose, 0.300 M trehalose, 0.218 M ranose, 0.8 M glycerol, 0.400 M inositol and 0.400 M sorbitol. Subsequent steps were the same as in the previous experiment. Evaluation of dierent cryoprotectant solutions Harvested shoot apices from A. viridis were ®rst precultured on BM supplemented with 0.8 M glycerol for 3 d then incubated in dierent cryoprotectant solutions for 25 min at 0 8C. The cryoprotectant solutions were: (1) PVS1
Turner et al.ÐCryopreservation of Endangered Species (19 % w/v glycerol, 13 % w/v ethylene glycol, 13 % w/v propylene glycol, 6 % w/v DMSO in half-strength MS liquid medium 0.5 M sorbitol) (Uragami et al., 1989), (2) PVS2 (30 % w/v glycerol, 15 % w/v ethylene glycol and 15 % w/v DMSO in half-strength MS liquid medium 0.4 M sucrose) (Sakai et al., 1990), (3) Mod-1 PVS2 (30 % w/v glycerol, 15 % w/v ethylene glycol, 7.5 % w/v DMSO, 7.5 % propylene glycol in half-strength MS liquid 0.4 M sucrose), (4) Mod-2 PVS2 (30 % w/v glycerol, 15 % w/v ethylene glycol, 15 % propylene glycol in half-strength MS liquid medium 0.4 M sucrose), (5) PVS3 (50 % w/v glycerol, 50 % w/v sucrose in water) (Nishizawa et al., 1993), (6) Mod PVS3 (50 % w/v glycerol, 50 % w/v sucrose, 5 % DMSO in water) (Nishizawa et al., 1993). The material was then rinsed and placed on recovery medium, or cooled in LN, then warmed, rinsed and placed on recovery medium. Comparison between the previous protocol and the modi®ed protocol Using the optimal combination from the preculture duration experiment, preculture sugar/polyalcohol experiments and cryoprotectant solution experiment, all six study species were evaluated using this modi®ed protocol. Shoot apices were again evaluated after: (a) harvest ( placed directly onto recovery medium without any treatment); (b) preculture; (c) preculture, PVS2 exposure and rinsing; and (d) preculture, PVS2 exposure, LN immersion, warming and rinsing. In the modi®ed protocol, 0.8 M glycerol replaced 0.4 M sorbitol, 3 d preculture replaced 2, and shoot apices were exposed to Mod-1 PVS2 for 25 min at 0 8C. These results were then compared with results from the previous protocol of Turner et al. (2000). Evaluation of a loading stage For selected species following preculture, shoot apices were placed in 2 ml loading solution consisting of 2 M glycerol, plus half-strength MS liquid culture medium (BM) supplemented with 0.4 M sucrose (Matsumoto et al., 1994; Charoensub et al., 1999). Shoot apices were incubated in this medium for 20 min at room temperature before placing in Mod-1 PVS2. Shoot apices were then rinsed and placed on recovery medium or immersed in LN, warmed, rinsed and placed on recovery medium. Scoring of survival and regeneration Each experimental treatment was repeated three times (three replicates) and each replicate consisted of 15 shoot apices. Survival was determined as detectable growth of all or part of apical tissues 1±28 d after treatment. Recovery was determined as continued growth of apices into shoots. Data generated were analysed statistically by analysis of variance (ANOVA). The original data expressed as a prop portion ( %) were transformed (arcsin ) to conform to ANOVA assumptions. Mean comparisons were then made using Fisher's protected LSD, at the 95 % con®dence level.
373
R E S ULT S Responses of shoot apices ( for six dierent Haemodoraceae species) to dierent steps in the protocol The response of shoot apices to each step in the protocol was evaluated using the original protocol developed for A.viridis ssp. viridis by Turner et al. (2000). Across all six species there was very high survival prior to the incorporation of the preculture step (84.0±100.0 %) (data not shown) but survival decreased signi®cantly with the addition of a preculture stage, PVS2 exposure and LN immersion (P 5 0.05). Although some survival following LN immersion was recorded for all six species, only A. viridis ssp. terraspectans showed acceptable levels of survival (61.1 %) (data not shown). Shoot apices exposed to preculture or preculture and PVS2, but not immersed in LN, displayed more rapid growth rates (approximately twice those of shoot apices cooled with LN) (data not shown). This was observed for all species. Comparison of shoot apex responses for six dierent Haemodoraceae species exposed to PVS2 directly after excision For all six species, shoot apices that were excised and then incubated in PVS2 without any preculture treatments had very low survival (0±2 %) (data not shown), and all apices appeared `bleached' 2 to 3 d after treatment. Comparison of preculture duration time Shoot apices of A. viridis ssp. terraspectans exposed to 0 d preculture, treated with PVS2, and LN immersion showed no survival, while non-LN immersed shoot apices with the same preculture duration (0 d) had very low survival (3.0 %) (Fig. 1). However following 1 d preculture, survival of both treated (non-cooled) and treated/cooled shoot apices increased signi®cantly (P 5 0.05) (Fig. 1). For both treatments, survival peaked after 3 d preculture. However, for treated shoot apices (non-cooled) and treated/cooled shoot apices, preculture for 7 d signi®cantly reduced survival (P 5 0.05) compared to 3 d preculture. Comparison of dierent sugars and polyalcohols at the same molarity (0.4 M) When compared at the same molarity, most of the sugars and polyalcohols examined gave similar levels of survival; the highest survival was obtained with glycerol (77.6 %), while the trehalose treatment resulted in signi®cantly lower survival (34.0 %) than all other compounds examined (P 5 0.05) (Fig. 2A). Comparison of dierent sugars and polyalcohols with the same number of hydroxyl groups Molarity of the sugars in the preculture media was adjusted in this experiment to yield a similar total number of hydroxyl groups (14.448 1023) of all sugars and polyalcohols. The number of hydroxyl groups was based
374
Turner et al.ÐCryopreservation of Endangered Species 100
100 Treated Control LN Cooled 80
80 Percentage survival
Percentage survival
A
90
60
40
20
70 60 50 40 30 20
80
Preculture duration
Shoot apices precultured for 3 d on 0.8 M glycerol, then incubated in dierent vitri®cation solutions had very similar levels of survival for all the treated controls (non-cooled). However, following LN immersion, PVS1 treatment resulted in no survival, while PVS3 and modi®ed PVS3 solutions gave low survival (12.5 and 12.8 %) (Fig. 3). All shoot apices that did not survive became `bleached' 2±3 d after warming. Shoot survival was high following treatment with PVS2 and the two modi®ed PVS2 solutions ( plus LN immersion); the results were not signi®cantly dierent between these treatments but were signi®cantly higher than the other vitri®cation solutions. Comparison of shoot apex responses for six dierent Haemodoraceae species harvested after dierent steps in the modi®ed cryopreservation protocol Survival following LN immersion was signi®cantly improved for A. humilis (P 5 0.05) (71.8 %), A. kalbarriensis (18.4 %) A. viridis (80.3 %) and C. wonganensis
50 40 30 20
Trehalose
Sucrose
Raffinose
Glucose
Inositol
10 Sorbitol
Comparison of dierent vitri®cation solutions
60
Glycerol
on 0.4 M sorbitol (the optimal sorbitol concentration) (Turner et al., 2000). Lowest survival was recorded for trehalose and the polysaccharides; ranose and sucrose resulted in lower survival than the monosaccharide glucose, or the polyalcohols (sorbitol, inositol and glycerol). Highest survival was provided by the polyalcohol glycerol (83.0 %), while the two other polyalcohols, sorbitol and inositol, ranked second (Fig. 2B).
70 Percentage survival
F I G . 1. Comparison of percentage shoot apex survival for A. viridis ssp. terraspectans precultured for 0, 1, 2, 3 and 7 d on 0.4 M sorbitol, incubated in PVS2 for 25 min at 0 8C, then rinsed in 1 M sucrose solution or immersed in liquid nitrogen (LN), then warmed and rinsed in 1 M sucrose washing solution (vertical bars represent twice the s.e.).
B
90
7 Days
3 Days
2 Days
1 Day
0 Days
10 0
Sugar/polyalcohol F I G . 2. A, Percentage survival for A. viridis ssp. terraspectans precultured on seven dierent sugars and polyalcohols at the same molarity (0.4 M) for 3 d. B, Percentage survival for A. viridis ssp. terraspectans when precultured on seven dierent sugars and polyalcohols adjusted to provide the same number of hydroxyl groups (14.448 1023) for 3 d. Shoot apices were then incubated in PVS2 for 25 min at 0 8C, plunged into LN, then warmed and rinsed in 1 M sucrose solution (vertical bars represent twice the s.e.).
(67.3 %) using the modi®ed protocol rather than the protocol of Turner et al. (2000) (Fig. 4). Furthermore, for these species there was also increased survival at dierent stages in the protocol. However, C. dielsia and C. micrantha showed no signi®cant improvements between the old and new protocols (P 4 0.05) (Fig. 4). The addition of a loading step For A. humilis, A. kalbarriensis, A.viridis and C. wonganensis, where high survival was achieved up to the LN
Turner et al.ÐCryopreservation of Endangered Species Treated Control LN Cooled 100
Percentage survival
80
60
40
20
Mod PVS3 (5% DMSO)
PVS3
Mod PVS2 (15% Propandiol)
Mod PVS2 (7.5% Propandiol/7.5% DMSO)
PVS2
PVS1
0
F I G . 3. Percentage survival of A. viridis ssp. terraspectans shoot apices exposed to dierent vitri®cation solutions. Material was precultured for 3 d on 0.8 M glycerol, then incubated for 25 min at 0 8C in one of six dierent vitri®cation solutions (PVS1, PVS2, Mod-1 PVS2, Mod-2 PVS2, PVS3 and Mod PVS3), then rinsed in 1 M sucrose solution or immersed in LN, warmed and rinsed in 1 M sucrose solution (vertical bars represent twice the s.e.).
immersion stage, a loading phase was added. For three of these species post-LN survival was higher (P 5 0.05) with the use of a loading phase (Fig. 5). Furthermore, signs of survival were ®rst noticed 1±2 d earlier for the loaded samples, while the initial rate of growth for these plants was also more rapid (data not shown). This was observed for all species. DISCUSSION The results presented in this study demonstrate the ecacy of using an indicator species (in this case Anigozanthos viridis ssp. terraspectans) to optimize cryopreservation protocols that can later be successfully applied to other related species. A. viridis ssp. terraspectans was chosen as
375
the indicator species due to its high multiplication rate, and because the initial protocol was developed on the closely related species A. viridis ssp. viridis (Turner et al., 2000). In comparison, the other species in this study had low multiplication rates, or insucient numbers which impeded large scale experimentation. All species examined with the original protocol had postLN survival; however, survival was low for most species, indicating that the protocol was sub-optimal. This was con®rmed when the protocol steps were examined, with all species exhibiting a sharp decline in survival prior to LN immersion. Therefore, to increase post-LN survival, adjustments were needed to produce higher survival in the preculture and PVS incubation phases. The importance of preculture duration is obvious when shoot apices from all species were excised and incubated in PVS2 for 25 min without preculture. Four of the six species did not survive, and survival was only 2 % for the other two species. These observations suggest that shoot apices need sucient preculture (at least 24 h) to acquire osmotolerance to PVS2 prior to being plunged into LN. Furthermore, for A. viridis ssp. terraspectans an increasing trend in post-LN survival was seen from 1 to 3 d preculture on sorbitol. Optimal preculture duration may vary for dierent species. For example, Grevillea scapigera (Touchell, 1995) and Macropidia fuliginosa (another Haemodoraceae species) achieved maximum post-LN survival following 2 d preculture, while Hopkinsia anoectocolea, another native monocot species, had the highest post-LN survival (46.4 %) after preculturing shoot apices on 0.2 M sorbitol for 3 d before incubation in PVS2 for 25 mins at 0 8C (unpubl. obs.). Preculture on dierent sugars and polyalcohols of the same molarity resulted in similar survival patterns except for the trehalose treatment which produced signi®cantly lower survival. This was unexpected as trehalose has been extensively found in a diverse range of desiccation-tolerant organisms (Crowe and Crowe, 1986). However, its use in plant systems has been limited (Bhandal et al., 1985; Reed, 1996). Of the other sugars/polyalcohols examined, glycerol treatments resulted in higher survival both at the same molarity (0.4 M) and when used with the same number of hydroxyl groups (0.8 M) as sorbitol (Fig. 2), although this dierence was not signi®cant. When treatments contain the same number of hydroxyl groups, glycerol is present at a concentration of 0.8 M which is twice the concentration of sorbitol, the polyalcohol used in the original protocol. The original study on A. viridis ssp. viridis found that 0.6 M sorbitol resulted in signi®cantly lower levels of survival (Turner et al., 2000). The mode of superior action for glycerol is not clear. It is thought that the hydroxyl groups present in sugars/ polyalcohols interact by hydrogen bonding with membrane phospholipids, imparting membrane stabilization during dehydration (Crowe and Crowe, 1986). Phunchindawan et al. (1996) and Thammasiri (1999) achieved high survival using a mixture of glycerol and sucrose in the preculture medium for horseradish hairy root cultures (at 1.0 M) and for embryonic axes of jackfruit (at 0.5 M).
376
Turner et al.ÐCryopreservation of Endangered Species
80 60 40 20 0
B C Treatment
80 60 40 20
D
A
Percentage survival
60 40 20
B
C
80 60 40 20 0
D
A*
Percentage survival
Percentage survival
80 60 40 20
C
C
D
100
E
B
B*
Treatment
100
A
D
D
Treatment
0
C
100
C
A
B
Treatment
80
0
B
0 A
100 Percentage survival
100
A Percentage survival
Percentage survival
100
D
Treatment
F
80 60 40 20 0
A
B
C
D
Treatment
F I G . 4. Comparison of percentage shoot apex survival between the original and modi®ed protocol for A. humilis ssp. chrysanthus (A), A. kalbarriensis (B) and A. viridis ssp. terraspectans (C), C. dielsia ssp. teres (D), C. micrantha (E), and C. wonganensis (F) after dierent steps in the cryopreservation protocol: A, no treatment ( freshly excised shoot apices were placed directly onto recovery medium); B, preculture on 0.4 M sorbitol or 0.8 M glycerol for 2 or 3 d; C, preculture on 0.4 M sorbitol or 0.8 M glycerol for 2 or 3 d, then incubation in 25 min PVS2 or 25 min Mod-1 PVS2, then rinsed in 1 M sucrose solution; D, preculture on 0.4 M sorbitol or 0.8 M glycerol for 2 or 3 d, then incubation in 25 min PVS2 or 25 min Mod-1 PVS2, then immersion in LN and ®nally warmed and rinsed in 1 M sucrose solution (vertical bars represent twice the s.e.). h, original; F, modi®ed; *, no data.
Successful utilization of dierent plant vitri®cation solutions such as PVS1 (Uragami et al., 1989), PVS2 (Sakai et al., 1990) and PVS3 (Nishizawa et al., 1993) are reported in the literature, but PVS2 is the most commonly used (Charoensub et al., 1999; Thammasiri, 1999; Thinh et al., 1999; Sakai et al., 2000). Results achieved from the PVS experiment demonstrated that before LN immersion very high survival was achieved for apices vitri®ed in the vitri®cation solutions tested.
However, following LN immersion only the PVS2 and modi®ed PVS2 solutions resulted in high post-LN survival. In fact, plants cryopreserved in the three PVS2 and modi®ed PVS2 solutions had very similar survival (Fig. 3)Ðeven in the Mod-2 PVS2 solution without DMSO. The replacement component propylene glycol has been reported to have similar properties to DMSO in that cell membranes are highly permeable to it and it has a low molecular weight (Boucaud and Brison, 1995), but it has
Turner et al.ÐCryopreservation of Endangered Species Non-Loaded Loaded
100
Percentage survival
80
60
40
C. wonganensis
A. viridis
A. kalbarriensis
0
A. humilis
20
Species F I G . 5. Comparison of shoot apex survival between four dierent Haemodoraceae species without a loading stage (3 d preculture on 0.8 M glycerol, 25 min exposure to Mod-1 PVS2, then LN immersion, warming and rinsing in 1 M sucrose solution) and with 20 min loading in a 2 M glycerol and 0.4 M sucrose loading solution (3 d preculture on 0.8 M glycerol, loading in 2 M glycerol loading solution, 25 min exposure to Mod-1 PVS2, then LN immersion, warming and rinsing in 1 M sucrose solution) (vertical bars represent twice the s.e.).
377
exhibiting higher survival prior to being plunged into LN. Loading signi®cantly improved survival for all four species (A. humilis, A. kalbarriensis, A. viridis and C. wonganensis). Loading is reported to be very eective in inducing dehydration tolerance of shoot apices to PVS2 (Matsumoto et al., 1994; Charoensub et al., 1999; Sakai et al., 2000), though its mode of action is not well understood (Matsumoto et al., 1998; Sakai, 2000). It may act by concentrating cytosolic cryoprotectants accumulated during preculture. Its actions may also be due to the protective eect of plasmolysis (Sakai, 2000). More recently other authors such as Charoensub et al. (1999), Thinh et al. (1999), Lambardi et al. (2000), and Pennycooke and Towill (2000) signi®cantly increased post-LN recovery using a loading solution in vitri®cation protocols for temperate and tropical plants. The major component of the loading solution is 2 M glycerol, thus glycerol seems to be a signi®cant factor in achieving high post-LN survival. Results of this study suggest that high post-LN survival can be achieved with alterations to preculture duration, the use of glycerol in the preculture medium and the addition of a loading step to a cryopreservation protocol. Furthermore, we have demonstrated that cryoprotectant solutions with reduced amounts of DMSO (7.5 and 0 %) can impart similar post-LN survival when compared to PVS2. Finally, a more ecient protocol applicable to a number of species was developed by incorporation of these steps which ultimately increased post-LN survival. We anticipate that a similar approach as outlined in this study should help to facilitate the rapid implementation of cryopreservation programs and, in the process, enhance conservation of rare and endangered plants both in Australia and elsewhere. L I T E R AT U R E C I T E D
also been reported to have low toxicity (Boucaud and Brison, 1995), which may be critical to survival for species that are DMSO sensitive. The other cryoprotectant solutions evaluated have all been reported to be highly successful for certain species (Uragami et al., 1989; Nishizawa et al., 1993) but in this study no survival or extremely low post-LN survival was found when PVS1, PVS3 and modi®ed PVS3 were used. It is probable that these solutions may be eective under dierent experimental conditions such as prolonged or shorter exposure periods; these aspects were not investigated in this study. Modi®cations made to the protocol based on the results of the present study signi®cantly increased the survival of four species (all three Anigozanthos species and Conostylis wonganensis). This increased survival was obvious across all steps in the protocol highlighting the need to minimize the detrimental eects of treatments at all stages in a cryopreservation protocol, not simply the post-LN stage. For example, for these species, preculture with glycerol for 3 d and incubation in modi®ed PVS2 ultimately provides better cryoprotection than the previous protocol. The loading step between preculture and dehydration with PVS2 was examined with the four study species
Bajaj YPS. 1995. Cryopreservation of plant cell, tissue, organ culture for the conservation of germplasm and biodiversity. In: Bajaj YPS, ed. Biotechnology in agriculture and forestry 32. Cryopreservation of plant germplasm I. Berlin: Springer-Verlag, 3±47. Benson EE. 1999. Cryopreservation. In: Benson EE, ed. Plant conservation biotechnology. London: Taylor and Francis, 83±95. Bhandal IS, Hauptmann M, Widholm JM. 1985. Trehalose as cryoprotectant for the freeze preservation of carrot and tobacco cells. Plant Physiology 78: 430±432. Boucaud MT, Brison M. 1995. Cryopreservation of germplasm of walnut (Juglans species). In: Bajaj YPS, ed. Biotechnology in agriculture and forestry 32. Cryopreservation of plant germplasm I. Berlin: Springer-Verlag, 129±147. Brown A, Thomson-Dans C, Marchant N, eds. 1998. Western Australia's threatened ¯ora. Western Australia: Department of Conservation and Land Management. Charoensub R, Phansiri S, Sakai A, Yongmenitchai W. 1999. Cryopreservation of cassava in vitro-grown shoot tips cooled to ÿ1968C by vitri®cation. Cryo Letters 20: 89±94. Crowe JH, Crowe LM. 1986. Stabilisation of membranes in anhydrobiotic organisms. In: Leopold C, ed. Membranes, metabolism and dry organisms. Ithaca: Comstock Publishing Associates, 188±209. Engelmann F. 2000. Importance of cryopreservation for the conservation of plant genetic resources. In: Engelmann F, Takagi H, eds. Cryopreservation of tropical germplasm. Tsukuba: Japanese International Research Centre for Agricultural Sciences, 8±20. Hopper SD. 1993. Kangaroo paws and cats paws. Perth: Department of Conservation and Land Management, 81±87.
378
Turner et al.ÐCryopreservation of Endangered Species
Lambardi M, Fabbri A, Caccavale A. 2000. Cryopreservation of white poplar (Populus alba L.) by vitri®cation of in vitro-grown shoot tips. Plant Cell Reports 19: 213±218. Matsumoto T, Sakai A, Nako Y. 1998. A novel preculture for enhancing the survival of in vitro-grown meristems of wasabi (Wasabi japonica) cooled to ÿ1968C by vitri®cation. Cryo-Letters 19: 27±36. Matsumoto T, Sakai A, Yamada K. 1994. Cryopreservation of in vitrogrown apical meristems of wasabi (Wasabia japonica) by vitri®cation and subsequent high plant regeneration. Plant Cell Reports 13: 442±446. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiologia Plantarium 15: 473±497. Nishizawa S, Sakai A, Amano Y, Matsuzawa T. 1993. Cryopreservation of asparagus (Asparagus ocinalis L.) embryonic suspension cells and subsequent plant regeneration by vitri®cation. Plant Science 91: 67±73. Pennycooke JC, Towill LE. 2000. Cryopreservation of shoot tips from in vitro plants of sweet potato (Ipomoea batatas (L) Lam.) by vitri®cation. Plant Cell Reports 19: 733±737. Phunchindawan M, Hirata K, Sakai A, Miyamoto K. 1996. Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures. Plant Cell Reports 16: 469±473. Reed BM. 1996. Pre-treatment strategies for cryopreservation of plant tissues. In: Normah MN, Narimah MH, Clyde MM, eds. Techniques in in vitro conservation. Proceedings of the international workshop on in vitro conservation of plant genetic resources, July 4±6 1995, Kuala Lumpur. Universiti Kebangsaan Malaysia Publishers, 73±87.
Sakai A. 2000. Development of cryopreservation techniques. In: Engelmann F, Takagi H, eds. Cryopreservation of tropical germplasm. Tsukuba: Japanese International Research Centre for Agricultural Sciences, 1±7. Sakai A, Kobayashi S, Oiyama I. 1990. Cryopreservation of nucellar cells of naval orange (Citrus sinesis Osb. var. brasiliensis Tanaka) by vitri®cation. Plant Cell Reports 9: 30±33. Sakai A, Matsumoto T, Hirai D, Niino T. 2000. Newly developed encapsulation-dehydration protocol for plant cryopreservation. Cryo-Letters 21: 53±62. Thammasiri K. 1999. Cryopreservation of embryonic axes of jackfruit. Cryo-Letters 20: 21±28. Thinh NT, Takagi H, Yashima S. 1999. Cryopreservation of in vitro grown shoot tips of banana (Musa sp) by vitri®cation method. Cryo-Letters 20: 163±174. Touchell DH. 1995. Principles of Cryobiology for Conservation of Threatened Species. PhD thesis, University of Western Australia, Perth. Touchell DH, Dixon KW. 1999. In vitro preservation. In: Bowes BG, ed. A colour atlas of plant conservation and propagation. London: Manson Publishing Ltd, 108±118. Touchell DH, Turner SR, Bunn E, Dixon KW. 2001. Cryostorage of somatic tissues of endangered Australian species. In: Bajaj YPS, ed. Biotechnology in agriculture and forestry 32. Cryopreservation of plant germplasm II. Berlin: Springer-Verlag. Turner SR, Touchell DH, Dixon K, Tan B. 2000. Cryopreservation of Anigozanthos viridis ssp. viridis and related taxa from the south west of Western Australia. Australian Journal of Botany 48: 739±744. Uragami A, Sakai A, Nagai M, Takahash T. 1989. Survival of cells and somatic embryos of Asparagus ocinalis cryopreserved by vitri®cation. Plant Cell Reports 8: 418±421.
Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modi®ed Vitri®cation Protocol S . R . T U R N E R * {{, T. S E N A R AT N A{, E . B U N N {, B . TA N {, K . W. D IX O N{ and D . H. TO U C HE L L} {Kings Park and Botanic Garden, West Perth, WA 6005, Australia, {Curtin University of Technology, Bentley, WA 6102, Australia and }USDA-ARS National Seed Storage Laboratory, 1111 South Mason St, Fort Collins, Colorado 80521, USA Received: 15 September 2000 Returned for revision: 3 November 2000 Accepted: 21 November 2000 Published electronically: 26 January 2001
An ecient vitri®cation procedure was developed and successfully applied to cryopreserve six endangered West Australian species ( family Haemodoraceae: Anigozanthos humilis ssp. chrysanthus Hopper; A. kalbarriensis Hopper; A. viridis ssp. terraspectans Hopper; Conostylis dielsia ssp. teres Hopper; C. micrantha Hopper and C. wonganensis Hopper). Species were initially evaluated for cryostorage using a basic vitri®cation protocol involving: culturing plantlets in vitro for 21 d; excision of shoot apices; preculture of apical tips on 0.4 M sorbitol for 2 d, followed by incubation in PVS2 ( plant vitri®cation solution 2) for 25 min at 0 8C, then direct immersion in liquid nitrogen (LN). Warming of retrieved material was for 1 min in a 40 8C water bath. Using this protocol ®ve of the six species exhibited low post-storage survival, while the sixth species, A. viridis ssp. terraspectans posted higher survival (61.1 %). Using A. viridis ssp. terraspectans as an indicator species, the initial protocol was modi®ed to include: 3 d preculture on 0.80 M glycerol, loading treatment with 2.0 M glycerol plus 0.4 M sucrose solution for 20 min, followed by 25 min exposure to a modi®ed PVS2. Survival was signi®cantly improved in the test species, and in further experiments three other species also showed signi®cant improvements with the new protocol. Key ®ndings include: eectiveness of glycerol in the preculture medium; the eect of preculture duration; the importance of a loading stage for these species; and the successful use of modi®ed PVS2 solutions with reduced or zero dimethyl sulfoxide # 2001 Annals of Botany Company (DMSO). Key words: A. humilis ssp. chrysanthus, A. kalbarriensis, A. viridis ssp. terraspectans, Conostylis dielsia ssp. teres, C. micrantha, C. wonganensis, kangaroo paws, Haemodoraceae, vitri®cation, cryopreservation, rare and endangered, conservation.
I N T RO D U C T I O N The Haemodoraceae (or `bloodroots') have representative taxa on several continents, including Africa, South America and North America (Hopper, 1993). In the south west of Western Australia (WA) this family shows considerable diversi®cation with 77 species belonging to seven genera. The majority of these genera i.e. Macropidia (black kangaroo paw), Anigozanthos (kangaroo paws), Blancoa, Conostylis, Phlebocarya and Tribonanthes are endemic to this region (Hopper, 1993). Amongst the endemic species of WA Haemodoraceae, many are classi®ed as rare and endangered, including seven species of Conostylis and three subspecies of Anigozanthos (Brown et al., 1998). Cryopreservation is currently being developed by agencies such as Kings Park and Botanic Garden (West Perth, Australia) to conserve threatened species. The advantages of cryostorage over conventional forms of germplasm storage include: reduction in maintenance costs associated with living collections; reduction in storage space; minimization of losses due to diseases and contamination (with living collections); and minimization of somaclonal variation (Bajaj, 1995). * For correspondence. E-mail [email protected]
0305-7364/01/030371+08 $35.00/00
For cryopreservation of endangered species, shoot apices are the preferred tissue source as they allow for the conservation of genetically stable tissues, which are essential for future re-introduction programs. Furthermore, cells found in the apical region of shoot apices are much more amenable to cryostorage protocols as they consist of small thin-walled actively dividing cells that have small vacuoles and a high nucleo-cytoplasmic ratio (Engelmann, 2000). Among several cryopreservation protocols, vitri®cation is a relatively simple method requiring no special cooling equipment (Benson, 1999). Most vitri®cation protocols utilized are based on plant vitri®cation solution 2 (PVS2) developed by Sakai et al. (1990), which involve the following steps: preculturing shoot apices on a sucrose enriched medium, loading treatment and dehydration with PVS2 prior to a plunge into liquid nitrogen (LN). Previously developed protocols for endangered Australian species involved preculturing shoot apices on a medium containing sorbitol, followed by incubation in PVS2 before direct immersion in LN (Touchell and Dixon, 1999; Touchell et al., 2001). In a previous study, Turner et al. (2000) achieved postLN survival of 36 % for Anigozanthos viridis ssp. viridis when shoot apices were precultured on a 0.4 M sorbitol # 2001 Annals of Botany Company
372
Turner et al.ÐCryopreservation of Endangered Species
medium for 2 d, before exposure to PVS2 for 25 min, followed by direct immersion in LN. This protocol was also found to be applicable to other Haemodoraceae species, although survival in all taxa tested was very low. The objectives of this study were to examine post-LN survival of Haemodoraceae species in response to: (a) alterations to preculture duration; (b) sugars/polyalcohols in the preculture medium; (c) vitri®cation solutions; and (d) the addition of a loading step. M AT E R I A L S A N D M E T H O D S
using a 0.2 mM Acrodisc1 32 ®lter prior to use. Treated shoot apices were then washed three times for 4 min per rinse, in half-strength MS liquid basal medium containing 1.0 M sucrose. After washing, shoot apices were placed onto recovery medium to assess the survival prior to LN immersion. Apices to be cooled were harvested, precultured, incubated in PVS2 as described above, plunged into LN and stored for at least 30 min prior to warming in a 40 8C water bath for 1 min. After warming, shoot apices were removed from the PVS2 medium and washed as described previously. After washing, shoot apices were placed onto recovery medium.
Species and in vitro culture The six species used in this study were: Conostylis dielsia ssp. teres, C. micrantha, C. wonganensis, Anigozanthos viridis ssp. terraspectans, A. humilis ssp. chrysanthus and A. kalbarriensis. With the exception of A. humilis ssp. chrysanthus, all species were obtained from in vitro shoot culture stocks maintained at the Plant Science Laboratory, Kings Park and Botanic Garden (Perth) and derived from ®eld collected shoots. In the case of A. humilis ssp. chrysanthus, shoot cultures were initiated from zygotic embryos. In vitro plantlets were subcultured at 3 to 4 weekly intervals on a basal medium (BM) containing half strength Murashige and Skoog (MS) salts (Murashige and Skoog, 1962) supplemented with 3.0 mM thiamine hydrochloride, 2.5 mM pyridoxine hydrochloride, 4.0 mM niacin, 0.5 mM MES buer, 0.5 mM myo-inositol, 60 mM sucrose, 0.5 mM 6benzylaminopurine (BAP) and solidi®ed with 0.8 % (w/v) agar. The pH of the medium was adjusted to 5.9 prior to the addition of agar, then autoclaved at 121 8C for 20 min. Plant cultures were incubated at 22±25 8C and illuminated with 16 h light with photosynthetic photon ¯ux density of 30 mmol m ÿ2 s ÿ1. Evaluation of the previously developed protocol In the ®rst experiment the ecacy of the protocol developed by Turner et al. (2000) was evaluated using the six study species. Turner et al.'s protocol consisted of six phases: culture, preculture, PVS2 exposure, LN immersion, warming and recovery. Shoot apices were harvested and evaluated following four key stages in the protocol: (a) immediately after harvest ( placed directly onto recovery medium without any treatment); (b) after preculture; (c) after preculture and PVS2 exposure; and (d) after preculture, PVS2 exposure, LN immersion, warming and washing. In the preculture stage, shoot apices 1.0±1.5 mm long which were excised from 19±23-d-old shoots were precultured on BM supplemented with 0.4 M sorbitol for 2 d. These shoot apices were then placed onto BM supplemented with 2 mM choline chloride recovery medium (in darkness for 7 d) (Touchell et al., 2001), with or without incubation in PVS2 (and for a sub-sample, LN immersion). Shoot apices at this stage were exposed to PVS2 at 0 8C for 25 min. PVS2 comprised 30 % w/v glycerol, 15 % w/v ethylene glycol and 15 % w/v dimethyl sulfoxide (DMSO) in half-strength MS liquid medium supplemented with 0.4 M sucrose (Sakai et al., 1990). The medium was ®lter-sterilized
Comparison of shoot apex responses ( for six dierent Haemodoraceae species) after direct exposure to PVS2 Shoot apices were harvested from all species and immediately incubated in PVS2. These apices were then rinsed in 1 M sucrose solution and placed on recovery medium. Subsequent experiments were designed to modify and improve the above protocol. Comparison of preculture duration time Shoot apices of A. viridis were precultured for 0, 1, 2, 3 and 7 d on BM containing 0.4 M sorbitol, incubated in PVS2 at 0 8C, washed and placed on recovery medium (without placing in LN) or plunged directly into LN, warmed, washed, and placed on recovery medium. Comparison of dierent sugars and polyalcohols in the preculture medium Seven dierent sugars and polyalcohols were evaluated in preculture media using Anigozanthos viridis shoot apices. In the ®rst experiment, shoot apices were precultured on BM supplemented with either 0.4 M glucose, sucrose, trehalose, ranose, glycerol, inositol or sorbitol for 3 d, then incubated in PVS2, cooled in LN, warmed and placed on recovery medium as noted above. In the second experiment, shoot apices were placed on BM supplemented with dierent molarities of these compounds. Molarities were formulated to provide the same equimolar concentrations of hydroxyl (OH) groups as 0.4 M sorbitol (14.448 1023 l ÿ1 of preculture medium). This was formulated by taking into account the total number of OH groups per molecule (e.g. sorbitol has six, glycerol has three) then adjusting the concentration to provide the same number of OH groups as 0.4 M sorbitol. Using this procedure the following concentrations were determined: 0.480 M glucose, 0.300 M sucrose, 0.300 M trehalose, 0.218 M ranose, 0.8 M glycerol, 0.400 M inositol and 0.400 M sorbitol. Subsequent steps were the same as in the previous experiment. Evaluation of dierent cryoprotectant solutions Harvested shoot apices from A. viridis were ®rst precultured on BM supplemented with 0.8 M glycerol for 3 d then incubated in dierent cryoprotectant solutions for 25 min at 0 8C. The cryoprotectant solutions were: (1) PVS1
Turner et al.ÐCryopreservation of Endangered Species (19 % w/v glycerol, 13 % w/v ethylene glycol, 13 % w/v propylene glycol, 6 % w/v DMSO in half-strength MS liquid medium 0.5 M sorbitol) (Uragami et al., 1989), (2) PVS2 (30 % w/v glycerol, 15 % w/v ethylene glycol and 15 % w/v DMSO in half-strength MS liquid medium 0.4 M sucrose) (Sakai et al., 1990), (3) Mod-1 PVS2 (30 % w/v glycerol, 15 % w/v ethylene glycol, 7.5 % w/v DMSO, 7.5 % propylene glycol in half-strength MS liquid 0.4 M sucrose), (4) Mod-2 PVS2 (30 % w/v glycerol, 15 % w/v ethylene glycol, 15 % propylene glycol in half-strength MS liquid medium 0.4 M sucrose), (5) PVS3 (50 % w/v glycerol, 50 % w/v sucrose in water) (Nishizawa et al., 1993), (6) Mod PVS3 (50 % w/v glycerol, 50 % w/v sucrose, 5 % DMSO in water) (Nishizawa et al., 1993). The material was then rinsed and placed on recovery medium, or cooled in LN, then warmed, rinsed and placed on recovery medium. Comparison between the previous protocol and the modi®ed protocol Using the optimal combination from the preculture duration experiment, preculture sugar/polyalcohol experiments and cryoprotectant solution experiment, all six study species were evaluated using this modi®ed protocol. Shoot apices were again evaluated after: (a) harvest ( placed directly onto recovery medium without any treatment); (b) preculture; (c) preculture, PVS2 exposure and rinsing; and (d) preculture, PVS2 exposure, LN immersion, warming and rinsing. In the modi®ed protocol, 0.8 M glycerol replaced 0.4 M sorbitol, 3 d preculture replaced 2, and shoot apices were exposed to Mod-1 PVS2 for 25 min at 0 8C. These results were then compared with results from the previous protocol of Turner et al. (2000). Evaluation of a loading stage For selected species following preculture, shoot apices were placed in 2 ml loading solution consisting of 2 M glycerol, plus half-strength MS liquid culture medium (BM) supplemented with 0.4 M sucrose (Matsumoto et al., 1994; Charoensub et al., 1999). Shoot apices were incubated in this medium for 20 min at room temperature before placing in Mod-1 PVS2. Shoot apices were then rinsed and placed on recovery medium or immersed in LN, warmed, rinsed and placed on recovery medium. Scoring of survival and regeneration Each experimental treatment was repeated three times (three replicates) and each replicate consisted of 15 shoot apices. Survival was determined as detectable growth of all or part of apical tissues 1±28 d after treatment. Recovery was determined as continued growth of apices into shoots. Data generated were analysed statistically by analysis of variance (ANOVA). The original data expressed as a prop portion ( %) were transformed (arcsin ) to conform to ANOVA assumptions. Mean comparisons were then made using Fisher's protected LSD, at the 95 % con®dence level.
373
R E S ULT S Responses of shoot apices ( for six dierent Haemodoraceae species) to dierent steps in the protocol The response of shoot apices to each step in the protocol was evaluated using the original protocol developed for A.viridis ssp. viridis by Turner et al. (2000). Across all six species there was very high survival prior to the incorporation of the preculture step (84.0±100.0 %) (data not shown) but survival decreased signi®cantly with the addition of a preculture stage, PVS2 exposure and LN immersion (P 5 0.05). Although some survival following LN immersion was recorded for all six species, only A. viridis ssp. terraspectans showed acceptable levels of survival (61.1 %) (data not shown). Shoot apices exposed to preculture or preculture and PVS2, but not immersed in LN, displayed more rapid growth rates (approximately twice those of shoot apices cooled with LN) (data not shown). This was observed for all species. Comparison of shoot apex responses for six dierent Haemodoraceae species exposed to PVS2 directly after excision For all six species, shoot apices that were excised and then incubated in PVS2 without any preculture treatments had very low survival (0±2 %) (data not shown), and all apices appeared `bleached' 2 to 3 d after treatment. Comparison of preculture duration time Shoot apices of A. viridis ssp. terraspectans exposed to 0 d preculture, treated with PVS2, and LN immersion showed no survival, while non-LN immersed shoot apices with the same preculture duration (0 d) had very low survival (3.0 %) (Fig. 1). However following 1 d preculture, survival of both treated (non-cooled) and treated/cooled shoot apices increased signi®cantly (P 5 0.05) (Fig. 1). For both treatments, survival peaked after 3 d preculture. However, for treated shoot apices (non-cooled) and treated/cooled shoot apices, preculture for 7 d signi®cantly reduced survival (P 5 0.05) compared to 3 d preculture. Comparison of dierent sugars and polyalcohols at the same molarity (0.4 M) When compared at the same molarity, most of the sugars and polyalcohols examined gave similar levels of survival; the highest survival was obtained with glycerol (77.6 %), while the trehalose treatment resulted in signi®cantly lower survival (34.0 %) than all other compounds examined (P 5 0.05) (Fig. 2A). Comparison of dierent sugars and polyalcohols with the same number of hydroxyl groups Molarity of the sugars in the preculture media was adjusted in this experiment to yield a similar total number of hydroxyl groups (14.448 1023) of all sugars and polyalcohols. The number of hydroxyl groups was based
374
Turner et al.ÐCryopreservation of Endangered Species 100
100 Treated Control LN Cooled 80
80 Percentage survival
Percentage survival
A
90
60
40
20
70 60 50 40 30 20
80
Preculture duration
Shoot apices precultured for 3 d on 0.8 M glycerol, then incubated in dierent vitri®cation solutions had very similar levels of survival for all the treated controls (non-cooled). However, following LN immersion, PVS1 treatment resulted in no survival, while PVS3 and modi®ed PVS3 solutions gave low survival (12.5 and 12.8 %) (Fig. 3). All shoot apices that did not survive became `bleached' 2±3 d after warming. Shoot survival was high following treatment with PVS2 and the two modi®ed PVS2 solutions ( plus LN immersion); the results were not signi®cantly dierent between these treatments but were signi®cantly higher than the other vitri®cation solutions. Comparison of shoot apex responses for six dierent Haemodoraceae species harvested after dierent steps in the modi®ed cryopreservation protocol Survival following LN immersion was signi®cantly improved for A. humilis (P 5 0.05) (71.8 %), A. kalbarriensis (18.4 %) A. viridis (80.3 %) and C. wonganensis
50 40 30 20
Trehalose
Sucrose
Raffinose
Glucose
Inositol
10 Sorbitol
Comparison of dierent vitri®cation solutions
60
Glycerol
on 0.4 M sorbitol (the optimal sorbitol concentration) (Turner et al., 2000). Lowest survival was recorded for trehalose and the polysaccharides; ranose and sucrose resulted in lower survival than the monosaccharide glucose, or the polyalcohols (sorbitol, inositol and glycerol). Highest survival was provided by the polyalcohol glycerol (83.0 %), while the two other polyalcohols, sorbitol and inositol, ranked second (Fig. 2B).
70 Percentage survival
F I G . 1. Comparison of percentage shoot apex survival for A. viridis ssp. terraspectans precultured for 0, 1, 2, 3 and 7 d on 0.4 M sorbitol, incubated in PVS2 for 25 min at 0 8C, then rinsed in 1 M sucrose solution or immersed in liquid nitrogen (LN), then warmed and rinsed in 1 M sucrose washing solution (vertical bars represent twice the s.e.).
B
90
7 Days
3 Days
2 Days
1 Day
0 Days
10 0
Sugar/polyalcohol F I G . 2. A, Percentage survival for A. viridis ssp. terraspectans precultured on seven dierent sugars and polyalcohols at the same molarity (0.4 M) for 3 d. B, Percentage survival for A. viridis ssp. terraspectans when precultured on seven dierent sugars and polyalcohols adjusted to provide the same number of hydroxyl groups (14.448 1023) for 3 d. Shoot apices were then incubated in PVS2 for 25 min at 0 8C, plunged into LN, then warmed and rinsed in 1 M sucrose solution (vertical bars represent twice the s.e.).
(67.3 %) using the modi®ed protocol rather than the protocol of Turner et al. (2000) (Fig. 4). Furthermore, for these species there was also increased survival at dierent stages in the protocol. However, C. dielsia and C. micrantha showed no signi®cant improvements between the old and new protocols (P 4 0.05) (Fig. 4). The addition of a loading step For A. humilis, A. kalbarriensis, A.viridis and C. wonganensis, where high survival was achieved up to the LN
Turner et al.ÐCryopreservation of Endangered Species Treated Control LN Cooled 100
Percentage survival
80
60
40
20
Mod PVS3 (5% DMSO)
PVS3
Mod PVS2 (15% Propandiol)
Mod PVS2 (7.5% Propandiol/7.5% DMSO)
PVS2
PVS1
0
F I G . 3. Percentage survival of A. viridis ssp. terraspectans shoot apices exposed to dierent vitri®cation solutions. Material was precultured for 3 d on 0.8 M glycerol, then incubated for 25 min at 0 8C in one of six dierent vitri®cation solutions (PVS1, PVS2, Mod-1 PVS2, Mod-2 PVS2, PVS3 and Mod PVS3), then rinsed in 1 M sucrose solution or immersed in LN, warmed and rinsed in 1 M sucrose solution (vertical bars represent twice the s.e.).
immersion stage, a loading phase was added. For three of these species post-LN survival was higher (P 5 0.05) with the use of a loading phase (Fig. 5). Furthermore, signs of survival were ®rst noticed 1±2 d earlier for the loaded samples, while the initial rate of growth for these plants was also more rapid (data not shown). This was observed for all species. DISCUSSION The results presented in this study demonstrate the ecacy of using an indicator species (in this case Anigozanthos viridis ssp. terraspectans) to optimize cryopreservation protocols that can later be successfully applied to other related species. A. viridis ssp. terraspectans was chosen as
375
the indicator species due to its high multiplication rate, and because the initial protocol was developed on the closely related species A. viridis ssp. viridis (Turner et al., 2000). In comparison, the other species in this study had low multiplication rates, or insucient numbers which impeded large scale experimentation. All species examined with the original protocol had postLN survival; however, survival was low for most species, indicating that the protocol was sub-optimal. This was con®rmed when the protocol steps were examined, with all species exhibiting a sharp decline in survival prior to LN immersion. Therefore, to increase post-LN survival, adjustments were needed to produce higher survival in the preculture and PVS incubation phases. The importance of preculture duration is obvious when shoot apices from all species were excised and incubated in PVS2 for 25 min without preculture. Four of the six species did not survive, and survival was only 2 % for the other two species. These observations suggest that shoot apices need sucient preculture (at least 24 h) to acquire osmotolerance to PVS2 prior to being plunged into LN. Furthermore, for A. viridis ssp. terraspectans an increasing trend in post-LN survival was seen from 1 to 3 d preculture on sorbitol. Optimal preculture duration may vary for dierent species. For example, Grevillea scapigera (Touchell, 1995) and Macropidia fuliginosa (another Haemodoraceae species) achieved maximum post-LN survival following 2 d preculture, while Hopkinsia anoectocolea, another native monocot species, had the highest post-LN survival (46.4 %) after preculturing shoot apices on 0.2 M sorbitol for 3 d before incubation in PVS2 for 25 mins at 0 8C (unpubl. obs.). Preculture on dierent sugars and polyalcohols of the same molarity resulted in similar survival patterns except for the trehalose treatment which produced signi®cantly lower survival. This was unexpected as trehalose has been extensively found in a diverse range of desiccation-tolerant organisms (Crowe and Crowe, 1986). However, its use in plant systems has been limited (Bhandal et al., 1985; Reed, 1996). Of the other sugars/polyalcohols examined, glycerol treatments resulted in higher survival both at the same molarity (0.4 M) and when used with the same number of hydroxyl groups (0.8 M) as sorbitol (Fig. 2), although this dierence was not signi®cant. When treatments contain the same number of hydroxyl groups, glycerol is present at a concentration of 0.8 M which is twice the concentration of sorbitol, the polyalcohol used in the original protocol. The original study on A. viridis ssp. viridis found that 0.6 M sorbitol resulted in signi®cantly lower levels of survival (Turner et al., 2000). The mode of superior action for glycerol is not clear. It is thought that the hydroxyl groups present in sugars/ polyalcohols interact by hydrogen bonding with membrane phospholipids, imparting membrane stabilization during dehydration (Crowe and Crowe, 1986). Phunchindawan et al. (1996) and Thammasiri (1999) achieved high survival using a mixture of glycerol and sucrose in the preculture medium for horseradish hairy root cultures (at 1.0 M) and for embryonic axes of jackfruit (at 0.5 M).
376
Turner et al.ÐCryopreservation of Endangered Species
80 60 40 20 0
B C Treatment
80 60 40 20
D
A
Percentage survival
60 40 20
B
C
80 60 40 20 0
D
A*
Percentage survival
Percentage survival
80 60 40 20
C
C
D
100
E
B
B*
Treatment
100
A
D
D
Treatment
0
C
100
C
A
B
Treatment
80
0
B
0 A
100 Percentage survival
100
A Percentage survival
Percentage survival
100
D
Treatment
F
80 60 40 20 0
A
B
C
D
Treatment
F I G . 4. Comparison of percentage shoot apex survival between the original and modi®ed protocol for A. humilis ssp. chrysanthus (A), A. kalbarriensis (B) and A. viridis ssp. terraspectans (C), C. dielsia ssp. teres (D), C. micrantha (E), and C. wonganensis (F) after dierent steps in the cryopreservation protocol: A, no treatment ( freshly excised shoot apices were placed directly onto recovery medium); B, preculture on 0.4 M sorbitol or 0.8 M glycerol for 2 or 3 d; C, preculture on 0.4 M sorbitol or 0.8 M glycerol for 2 or 3 d, then incubation in 25 min PVS2 or 25 min Mod-1 PVS2, then rinsed in 1 M sucrose solution; D, preculture on 0.4 M sorbitol or 0.8 M glycerol for 2 or 3 d, then incubation in 25 min PVS2 or 25 min Mod-1 PVS2, then immersion in LN and ®nally warmed and rinsed in 1 M sucrose solution (vertical bars represent twice the s.e.). h, original; F, modi®ed; *, no data.
Successful utilization of dierent plant vitri®cation solutions such as PVS1 (Uragami et al., 1989), PVS2 (Sakai et al., 1990) and PVS3 (Nishizawa et al., 1993) are reported in the literature, but PVS2 is the most commonly used (Charoensub et al., 1999; Thammasiri, 1999; Thinh et al., 1999; Sakai et al., 2000). Results achieved from the PVS experiment demonstrated that before LN immersion very high survival was achieved for apices vitri®ed in the vitri®cation solutions tested.
However, following LN immersion only the PVS2 and modi®ed PVS2 solutions resulted in high post-LN survival. In fact, plants cryopreserved in the three PVS2 and modi®ed PVS2 solutions had very similar survival (Fig. 3)Ðeven in the Mod-2 PVS2 solution without DMSO. The replacement component propylene glycol has been reported to have similar properties to DMSO in that cell membranes are highly permeable to it and it has a low molecular weight (Boucaud and Brison, 1995), but it has
Turner et al.ÐCryopreservation of Endangered Species Non-Loaded Loaded
100
Percentage survival
80
60
40
C. wonganensis
A. viridis
A. kalbarriensis
0
A. humilis
20
Species F I G . 5. Comparison of shoot apex survival between four dierent Haemodoraceae species without a loading stage (3 d preculture on 0.8 M glycerol, 25 min exposure to Mod-1 PVS2, then LN immersion, warming and rinsing in 1 M sucrose solution) and with 20 min loading in a 2 M glycerol and 0.4 M sucrose loading solution (3 d preculture on 0.8 M glycerol, loading in 2 M glycerol loading solution, 25 min exposure to Mod-1 PVS2, then LN immersion, warming and rinsing in 1 M sucrose solution) (vertical bars represent twice the s.e.).
377
exhibiting higher survival prior to being plunged into LN. Loading signi®cantly improved survival for all four species (A. humilis, A. kalbarriensis, A. viridis and C. wonganensis). Loading is reported to be very eective in inducing dehydration tolerance of shoot apices to PVS2 (Matsumoto et al., 1994; Charoensub et al., 1999; Sakai et al., 2000), though its mode of action is not well understood (Matsumoto et al., 1998; Sakai, 2000). It may act by concentrating cytosolic cryoprotectants accumulated during preculture. Its actions may also be due to the protective eect of plasmolysis (Sakai, 2000). More recently other authors such as Charoensub et al. (1999), Thinh et al. (1999), Lambardi et al. (2000), and Pennycooke and Towill (2000) signi®cantly increased post-LN recovery using a loading solution in vitri®cation protocols for temperate and tropical plants. The major component of the loading solution is 2 M glycerol, thus glycerol seems to be a signi®cant factor in achieving high post-LN survival. Results of this study suggest that high post-LN survival can be achieved with alterations to preculture duration, the use of glycerol in the preculture medium and the addition of a loading step to a cryopreservation protocol. Furthermore, we have demonstrated that cryoprotectant solutions with reduced amounts of DMSO (7.5 and 0 %) can impart similar post-LN survival when compared to PVS2. Finally, a more ecient protocol applicable to a number of species was developed by incorporation of these steps which ultimately increased post-LN survival. We anticipate that a similar approach as outlined in this study should help to facilitate the rapid implementation of cryopreservation programs and, in the process, enhance conservation of rare and endangered plants both in Australia and elsewhere. L I T E R AT U R E C I T E D
also been reported to have low toxicity (Boucaud and Brison, 1995), which may be critical to survival for species that are DMSO sensitive. The other cryoprotectant solutions evaluated have all been reported to be highly successful for certain species (Uragami et al., 1989; Nishizawa et al., 1993) but in this study no survival or extremely low post-LN survival was found when PVS1, PVS3 and modi®ed PVS3 were used. It is probable that these solutions may be eective under dierent experimental conditions such as prolonged or shorter exposure periods; these aspects were not investigated in this study. Modi®cations made to the protocol based on the results of the present study signi®cantly increased the survival of four species (all three Anigozanthos species and Conostylis wonganensis). This increased survival was obvious across all steps in the protocol highlighting the need to minimize the detrimental eects of treatments at all stages in a cryopreservation protocol, not simply the post-LN stage. For example, for these species, preculture with glycerol for 3 d and incubation in modi®ed PVS2 ultimately provides better cryoprotection than the previous protocol. The loading step between preculture and dehydration with PVS2 was examined with the four study species
Bajaj YPS. 1995. Cryopreservation of plant cell, tissue, organ culture for the conservation of germplasm and biodiversity. In: Bajaj YPS, ed. Biotechnology in agriculture and forestry 32. Cryopreservation of plant germplasm I. Berlin: Springer-Verlag, 3±47. Benson EE. 1999. Cryopreservation. In: Benson EE, ed. Plant conservation biotechnology. London: Taylor and Francis, 83±95. Bhandal IS, Hauptmann M, Widholm JM. 1985. Trehalose as cryoprotectant for the freeze preservation of carrot and tobacco cells. Plant Physiology 78: 430±432. Boucaud MT, Brison M. 1995. Cryopreservation of germplasm of walnut (Juglans species). In: Bajaj YPS, ed. Biotechnology in agriculture and forestry 32. Cryopreservation of plant germplasm I. Berlin: Springer-Verlag, 129±147. Brown A, Thomson-Dans C, Marchant N, eds. 1998. Western Australia's threatened ¯ora. Western Australia: Department of Conservation and Land Management. Charoensub R, Phansiri S, Sakai A, Yongmenitchai W. 1999. Cryopreservation of cassava in vitro-grown shoot tips cooled to ÿ1968C by vitri®cation. Cryo Letters 20: 89±94. Crowe JH, Crowe LM. 1986. Stabilisation of membranes in anhydrobiotic organisms. In: Leopold C, ed. Membranes, metabolism and dry organisms. Ithaca: Comstock Publishing Associates, 188±209. Engelmann F. 2000. Importance of cryopreservation for the conservation of plant genetic resources. In: Engelmann F, Takagi H, eds. Cryopreservation of tropical germplasm. Tsukuba: Japanese International Research Centre for Agricultural Sciences, 8±20. Hopper SD. 1993. Kangaroo paws and cats paws. Perth: Department of Conservation and Land Management, 81±87.
378
Turner et al.ÐCryopreservation of Endangered Species
Lambardi M, Fabbri A, Caccavale A. 2000. Cryopreservation of white poplar (Populus alba L.) by vitri®cation of in vitro-grown shoot tips. Plant Cell Reports 19: 213±218. Matsumoto T, Sakai A, Nako Y. 1998. A novel preculture for enhancing the survival of in vitro-grown meristems of wasabi (Wasabi japonica) cooled to ÿ1968C by vitri®cation. Cryo-Letters 19: 27±36. Matsumoto T, Sakai A, Yamada K. 1994. Cryopreservation of in vitrogrown apical meristems of wasabi (Wasabia japonica) by vitri®cation and subsequent high plant regeneration. Plant Cell Reports 13: 442±446. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiologia Plantarium 15: 473±497. Nishizawa S, Sakai A, Amano Y, Matsuzawa T. 1993. Cryopreservation of asparagus (Asparagus ocinalis L.) embryonic suspension cells and subsequent plant regeneration by vitri®cation. Plant Science 91: 67±73. Pennycooke JC, Towill LE. 2000. Cryopreservation of shoot tips from in vitro plants of sweet potato (Ipomoea batatas (L) Lam.) by vitri®cation. Plant Cell Reports 19: 733±737. Phunchindawan M, Hirata K, Sakai A, Miyamoto K. 1996. Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures. Plant Cell Reports 16: 469±473. Reed BM. 1996. Pre-treatment strategies for cryopreservation of plant tissues. In: Normah MN, Narimah MH, Clyde MM, eds. Techniques in in vitro conservation. Proceedings of the international workshop on in vitro conservation of plant genetic resources, July 4±6 1995, Kuala Lumpur. Universiti Kebangsaan Malaysia Publishers, 73±87.
Sakai A. 2000. Development of cryopreservation techniques. In: Engelmann F, Takagi H, eds. Cryopreservation of tropical germplasm. Tsukuba: Japanese International Research Centre for Agricultural Sciences, 1±7. Sakai A, Kobayashi S, Oiyama I. 1990. Cryopreservation of nucellar cells of naval orange (Citrus sinesis Osb. var. brasiliensis Tanaka) by vitri®cation. Plant Cell Reports 9: 30±33. Sakai A, Matsumoto T, Hirai D, Niino T. 2000. Newly developed encapsulation-dehydration protocol for plant cryopreservation. Cryo-Letters 21: 53±62. Thammasiri K. 1999. Cryopreservation of embryonic axes of jackfruit. Cryo-Letters 20: 21±28. Thinh NT, Takagi H, Yashima S. 1999. Cryopreservation of in vitro grown shoot tips of banana (Musa sp) by vitri®cation method. Cryo-Letters 20: 163±174. Touchell DH. 1995. Principles of Cryobiology for Conservation of Threatened Species. PhD thesis, University of Western Australia, Perth. Touchell DH, Dixon KW. 1999. In vitro preservation. In: Bowes BG, ed. A colour atlas of plant conservation and propagation. London: Manson Publishing Ltd, 108±118. Touchell DH, Turner SR, Bunn E, Dixon KW. 2001. Cryostorage of somatic tissues of endangered Australian species. In: Bajaj YPS, ed. Biotechnology in agriculture and forestry 32. Cryopreservation of plant germplasm II. Berlin: Springer-Verlag. Turner SR, Touchell DH, Dixon K, Tan B. 2000. Cryopreservation of Anigozanthos viridis ssp. viridis and related taxa from the south west of Western Australia. Australian Journal of Botany 48: 739±744. Uragami A, Sakai A, Nagai M, Takahash T. 1989. Survival of cells and somatic embryos of Asparagus ocinalis cryopreserved by vitri®cation. Plant Cell Reports 8: 418±421.