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Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification
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South African Journal of Botany 77 (2011) 397 – 403 www.elsevier.com/locate/sajb
Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification X.-L. Chen, J.-H. Li, X. Xin, Z.-E. Zhang, P.-P. Xin, X.-X. Lu⁎ Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China Received 24 March 2010; received in revised form 9 October 2010; accepted 13 October 2010
Abstract In this study, apical meristems from adventitious buds of three lily (Lilium L.) cultivars were successfully cryopreserved by dropletvitrification. The most effective techniques were as follows. Excised meristems from in vitro plantlets which had been sub-cultured for about 2 months were cold-hardened at 4 °C for 1 week, precultured on MS medium supplemented with 0.3 mol L−1 sucrose for 2 days, osmoprotected in loading solution for 20–40 min at room temperature and then soaked in PVS2 solution for 90–120 min at 0 °C, frozen in microdroplets of vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen. The meristems were then rapidly rewarmed by dilution solution, transferred to regeneration medium and stored in the dark for two weeks at 20 °C, and then cultured under white fluorescent light at an intensity of 2000 lux, with a 16 h photoperiod at 20 °C. The highest post-thaw survival percentages of three cultivars ‘Siberia’ (Lilium × siberia), Lilium lancifolium Thunb. and ‘Snow Queen’ Lilium × longiflorum were 65.0%, 83.8% and 43.3%, and regeneration percentages were 62.0%, 67.6% and 35.0%, respectively. The study demonstrated that cryopreservation by droplet-vitrification increased survival and regeneration percentages of certain lily cultivars compared with vitrification. Thus to cryopreserve lily meristems, droplet-vitrification method is preferable to the vitrification method. Crown Copyright © 2010 Published by Elsevier B.V. on behalf of SAAB. All rights reserved. Keywords: Apical meristem; Cryopreservation; Droplet-vitrification method; Lily (Lilium L); Orthogonal experimental design
1. Introduction Lilies comprise a genus (Lilium L. in Liliaceae, Monocotyledoneae) of 115 species, of which 55 species originated in China. The genus is broadly distributed over China and species grow in different geographical areas, such as L. dauricum in Heilongjiang, L. martagon var. pilosiusculum in Xinjiang and L. formosanum in Taiwan (Fu et al., 2002; Long et al., 1999). Lilies are perennial bulbous herbs and since they are vegetative propagated, the germplasm resources cannot be preserved in low temperature seed banks. In vivo field preservation is labour intensive and there is a considerable risk of loss due to disease or extreme weather. On the other hand, in vitro tissue culture conservation is susceptible to contamination and somaclonal variation. Cryopreservation is the best choice for conservation
⁎ Corresponding author. Tel.: +86 10 62174099; fax: +86 10 62114309. E-mail addresses: [email protected], [email protected] (X.-X. Lu).
of lily germplasm resources due to its safety, repeatability and long-term storage possibilities. In 1973, the cultured cells of carrot were successfully cryopreserved for the first time. In the past 30 years, remarkable progress in cryopreservation has been made. Some simplified and valuable cryogenic techniques, such as the vitrification method (Sakai et al., 1990), air-drying method (Uragami et al., 1990), encapsulation-dehydration method (Fabre and Dereuddre, 1990) and the droplet-freezing method (Mix-Wagner et al., 2000; Schäfer-Menuhr et al., 1997) have been developed. Some new methods based on the combination of the last two methods have also been developed, such as encapsulation-vitrification method (Hirai and Sakai, 1999), and droplet-vitrification method (Leunufna and Keller, 2003). The number of species that can be cryopreserved has increased dramatically in recent years. For successful cryopreservation, it is essential to avoid lethal intracellular freezing which can occur during rapid cooling in liquid nitrogen (Sakai, 1995). Thus the materials to be cryopreserved have to be sufficiently dehydrated to avoid the
0254-6299/$ - see front matter. Crown Copyright © 2010 Published by Elsevier B.V. on behalf of SAAB. All rights reserved. doi:10.1016/j.sajb.2010.10.005
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lethal effects of intracellular ice crystals formation. To induce dehydration tolerance, materials need some treatments before being plunged into liquid nitrogen (LN), such as preculture on medium with a high sucrose concentration, treatment in loading solution, and exposure to plant vitrification solution (PVS). The injurious effects caused by the dehydration process are reduced by optimizing the duration of every step. The cooling rate is also a key factor for successful cryopreservation, because the ultra rapid freezing rate helps to avoid intracellular freezing and thus obtain a vitrified state during freezing (Fahy et al., 1984). Lilies have been cryopreserved by encapsulation-dehydration and vitrification in several previous studies (Bouman et al., 2003; Chen et al., 2007; Matsumoto and Sakai, 1995; Matsumoto et al., 1995; Zhang et al., 2004). Here we report for the first time the successful cryopreservation of meristems from adventitious buds of lily by droplet-vitrification. We also compared differences in survival percentage and regeneration percentage after cryopreservation between the droplet-vitrification and vitrification methods. Factors affecting the survival percentage and regeneration percentage were analyzed in order to provide a foundation for long-term and large-scale cryopreservation of lily meristems. 2. Materials and methods 2.1. Plant material The tissue culture of lily cultivar ‘Siberia’ (Lilium × siberia) was mainly used in the present study. Stock cultures of other two cultivars, Lilium lancifolium Thunb. and ‘Snow Queen’ (Lilium × longiflorum) were also used for comparison. The basal medium used for all the trials was Murashige & Skoog (MS) medium containing 3% sucrose and solidified with 7 g L−1 agar, and adjusted to pH 5.8 prior to autoclaving at 121 °C for 15 min. Adventitious buds were formed from the surface of bulb-scale segments after 30 days on MS medium with 6-benzylaminopurine (BA) 1.5 mg L −1 , α-Naphthalene acetic acid (NAA) 0.1 mg L−1, 250 mg L−1 casein hydrolysate, 3% sucrose, and 7 g L−1 agar. Then the adventitious buds were sub-cultured on MS medium with 2.0 mg L−1 BA, NAA 0.1 mg L−1, 3% sucrose, and 7 g L−1 agar to multiply under white fluorescent light (2000 lx) with a 16 h day−1 photoperiod at 20 °C for about 45 days. After sub-culture, they were grown on solidified MS medium in sterilized glass flasks under white fluorescent light at an intensity of 2000 lx with a 16 h photoperiod at 20 °C. 2.2. Cold-hardening and preculture In vitro plantlets sub-cultured for about 2 months were coldhardened at 4 °C for 1 week under white fluorescent light at an intensity of 2000 lx with a 16 h photoperiod. Meristems of about 1–2 mm in diameter with one or two leaf primordia were dissected from the cold-hardened and non-hardened segments under a binocular microscope in axenic conditions. The apical meristems were precultured in liquid MS basal medium supplemented with different sucrose concentrations (0, 0.2,
0.3, 0.4 and 0.5 mol L−1) for different days (0, 1, 2, 3 and 5 days) under the same light condition as indicated earlier. 2.3. Loading and vitrification procedure Pre-cultured meristems were osmoprotected in loading solution for 20–40 min at room temperature. The loading solution contained MS basal medium supplemented with 2 mol L−1 glycerol and 0.4 mol L−1 sucrose. The osmoprotected meristems were transferred onto sterilized filter paper for 30 s in order to absorb the exterior loading solution. The meristems were then soaked in PVS2 solution at 0 °C for various lengths of time (0, 60, 90, 120, and 150 min). The PVS2 solution consisted of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) dimethyl sulfoxide (Me2SO), and 0.4 mol L−1 sucrose in MS solution ( Sakai et al., 1990). In the droplet-vitrification treatment, 5–6 meristems were transferred to approximately 15-μl droplets of PVS2 solution on thin strips of sterile aluminium foil. The aluminium foil strips were folded to enclose the shoot tips. The folded foil strips were then carefully immersed into liquid nitrogen (LN) using fine forceps. After immersion, the strips were quickly transferred to 2 mL cryotubes which were immediately plunged into LN. In the vitrification treatment, meristems were transferred into 2 mL cryotubes containing 1 mL fresh ice-cold PVS2 and then plunged into LN. 2.4. Thawing and PVS2 dilution Samples were maintained in LN for at least 1 day. The meristems on the foil strips were then rapidly thawed by being immersed into 10 mL of dilution solution (DS) at room temperature for 15 min. DS consisted MS medium with 1.2 mol L−1 sucrose. The meristems were unloaded from the foil strips. This step also rinsed the PVS2 from the meristems. After 15 min, meristems were then immersed in 10 mL fresh DS for another 15 min. In the vitrification method, cryotubes were rapidly thawed in a water bath at 40 °C for 80 s. The PVS2 solution in the cryotubes was drained by a Pasteur pipette and replaced with DS. After 15 min, meristems were then kept for another 15 min in 10 mL fresh DS. 2.5. Plant recovery and viability After dilution of PVS2, meristems were placed onto regeneration medium composed of MS supplemented with 0.5 mg L−1 BA, 0.l mg L−1 NAA, 0.3 mg L−1 Gibberellic acid (GA3), 30 g L−1 sucrose, and 7 g L−1 agar and stored in the dark for two weeks at 20 °C. They were then cultured under white fluorescent light at an intensity of 2000 lx, with a 16 h photoperiod at 20 °C. The survival percentage was recorded as percentage of meristems that remained green after being cultured for one day in daylight. The regeneration percentage was recorded as percentage of the meristems producing normal shoots 4 weeks after planting.
X.-L. Chen et al. / South African Journal of Botany 77 (2011) 397–403
2.6. Experimental design
399
loaded for 20–40 min at room temperature and dehydrated with PVS2 for 90–120 min at 0 °C.
Orthogonal tests were applied for the study of dropletvitrification. Four factors and three levels of each factor were used and are listed in Table 1. Sucrose concentration in the preculture medium, duration of preculture and PVS2 treatment were analyzed by one-factor analysis in which one-factor was changeable and the other factors determined by the optimal treatment obtained from the orthogonal analysis results described in the Results section. Ten to twelve meristems were tested for each of the three repetitions per experiment.
2.7. Statistical evaluation
3.2. Responses of meristems after cryopreservation Four features distinguished meristems after plating on regeneration media for two weeks: (i) shoot tips becoming white indicated immediate tissue death after being plunged into LN; (ii) completely or partially black shoot tips indicated that there were reactions following cryopreservation (e.g. production and oxidation of polyphenols); (iii) callusing of meristems; and (iv) shoot tip regeneration (Fig. 1A1–4). 3.3. Cold-hardening
Results are presented as mean percentages with their standard error, and evaluated by SPSS 11.5 and Excel software. Significance of difference between mean values was assessed by analysis of variance (ANOVA) with Duncan's multiple range test (P b 0.05).
3. Results
The effects of cold-hardening on the survival percentage and regeneration percentage were summarized in Table 2. Post-thaw survival percentage and regeneration percentage of cold-hardened meristems were significantly higher than those of controls (P b 0.05), indicating the desirability of coldhardening. 3.4. Sucrose concentration during preculture
3.1. Orthogonal analysis The results of the orthogonal analysis are shown in Table 1. Except for the factor of loading time, results among all levels of the other three factors were significantly different. The order of significance was sucrose concentration N PVS2 treatment time duration of preculture N loading time. The optimal procedure led to the following recommendations: apical meristems should be cold-hardened for 1 week at 4 °C, precultured on MS medium supplemented with 0.3 mol L−1 sucrose for 2–3 days,
The sucrose concentration in preculture media significantly affected the post-thaw survival and regeneration percentages (Fig. 2), which increased at first, and then decreased with further increase in sucrose concentration. At 0.3 mol L−1 sucrose, survival and regeneration percentages peaked, reaching 69.2% and 63.8%, respectively. Adding more sucrose above 0.3 mol L−1 decreased the overall survival and regeneration capacity. 3.5. Preculture time
Table 1 Orthogonal design⁎ and analysis results. Treatment no.
Sucrose concentration (mol/L)
Preculture time (days)
Loading time (min)
PVS2 treatment time (min)
Survival percentage (%)
1 2 3 4 5 6 7 8 9 T1 T2 T3
0.3 (1) 0.3 (1) 0.3 (1) 0.4 (2) 0.4 (2) 0.4 (2) 0.5 (3) 0.5 (3) 0.5 (3) 51.9 a 21.5 b 16.9 b
1 (1) 2 (2) 3 (3) 1 (1) 2 (2) 3 (3) 1 (1) 2 (2) 3 (3) 18.5 b 31.2 ab 40.6 a
20 (1) 30 (2) 40 (3) 30 (2) 40 (3) 20 (1) 40 (3) 20 (1) 30 (2) 28.7 a 34.2 a 27.4 a
60 (1) 90 (2) 120 (3) 120 (3) 60 (1) 90 (2) 90 (2) 120 (3) 60 (1) 18.5 b 45.7 a 26.0 ab
27.3 72.7 55.6 10 8.3 46.2 18.2 12.5 20
The numbers in brackets refer to treatment level. T1–3 indicate the average value of the same level within the same column. Data within the same column followed by different letters are significantly different at the 0.05 level (Duncan's Multiple Range Test). ⁎ The orthogonal design is applied to preliminarily determine the optimal treatment as the basis of one-factor analysis in which one-factor was changeable and other factors were determined by the optimal treatment obtained from the orthogonal analysis.
Based on the sucrose concentration results, meristems were precultured in media with 0.3 mol L−1 sucrose for various lengths of time. Preculture time significantly affected the postthaw survival and regeneration percentages (Fig. 3) which were highest, 70.1% and 62.9%, respectively, when preculture was for 2 days. Percentages were significantly higher than for meristems precultured for 1 day or for the control. Post-thaw survival and regeneration percentages decreased steadily when preculture was for more than 2 days. 3.6. Time of PVS2 treatment Survival and regeneration percentages increased with increased exposure time to PVS2 and reached their highest when meristems were dehydrated for 90 min, then decreasing with further exposure (Fig. 4). However, the reduced percentages at 120 min were not significantly lower than those at 90 min. When dehydrated for 150 min, the survival percentage and regeneration percentage decreased to 15.3% and 7.4%, respectively. No meristem survived cryopreservation without PVS2 treatment.
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Fig. 1. Meristem responses after cryopreservation. Four responses of meristems after cryopreservation are illustrated. (A-1) whitened meristem; (A-2) blackened meristem; (A-3) callus; and (A-4) shoot development after cryopreservation. (B) Regenerated plantlets 4 weeks after cryopreservation; (C) regenerated plantlets 8 weeks after cryopreservation; and (D) regenerated plantlet after cryopreservation compared with control (untreated control to the left).
3.7. Comparison of the vitrification method and the droplet-vitrification method The three lily cultivars, ‘Siberia’, Lilium lancifolium Thunb. and ‘Snow Queen’ were cryopreserved by the droplet-vitrification and vitrification methods, and survival and regeneration percentages were compared. The results are shown in Table 3. ‘Siberia’ and L. lancifolium Thunb. responded significantly differently (P b 0.05) to the two methods. The survival and regeneration percentages when using droplet-vitrification method were significantly higher than those of the vitrification method. However, ‘Snow Queen’ meristems did not show any significant difference (P N 0.05) between the two methods. 4. Discussion In this paper, we compared two cryopreservation methods of droplet-vitrification and vitrification. Matsumoto and Sakai
(1995) and Matsumoto et al. (1995) cryopreserved lily meristems by encapsulation-dehydration and vitrification in previous studies and the percentage of shoot formation was high, but when we used their vitrification protocol the regeneration percentage was not as high as that achieved by those authors. The differences may have been caused by any of several factors, including donor plant variety, status, and culture conditions. For example, Panis et al. (2005) cryopreserved banana meristems using the protocol of Thinh et al. (1999), but the regeneration percentage was far lower than that achieved (by Thinh). However, in our study, for two of the three varieties tested, cryopreservation by droplet-vitrification relatively increased survival and regeneration percentages compared with vitrification. The greatest difference between the two protocols is freezing rate. The cooling rate in dropletvitrification which is about 130 °C s−1 is faster than that for vitrification of about 6 °C s−1 (Towill and Bonnart, 2003). Fahy et al. (1984) suggested that a high cooling rate helps obtain a
Table 2 Effects of cold-hardening on survival percentage and regeneration percentage. Control
Cold-hardened
Types of df and mean square
df
Mean square
F
Sig.
Survival percentage (% ± S.E.)
31.0 ± 4.6
63.1 ± 6.8 60.1 ± 5.1
1548.827 100.427 1660.007 56.117
0.017
26.8 ± 3.3
1 4 1 4
15.422
Regeneration percentage (% ± S.E.)
Between groups Within groups Between groups Within groups
29.581
0.006
Material: ‘Siberia.’
X.-L. Chen et al. / South African Journal of Botany 77 (2011) 397–403
Regeneration
Survival 90 a
80
a
Percentage (%)
70 60
b b
50 40
c
30 20
c
c
c c
c
10 0
0
0.2
0.3
0.4
0.5
Sucrose concentration (M) Fig. 2. Effects of preculture media with different sucrose concentrations on the survival and regeneration percentages. Values are averages of three or four independent cryopreservation experiments. Error bars represent standard errors. Bars marked by the same letter are not significantly different for survival and regeneration percentages according to Duncan's Multiple Range Test. (P N 0.05).
vitrified state during freezing. Droplet-vitrification developed from the simple droplet-freezing method, which was originally applied to shoot tips of potato (Schäfer-Menuhr et al., 1997) and asparagus (Mix-Wagner et al., 2000) when the cryoprotectant solution was 10% dimethyl sulphoxide (Me2SO/DMSO). In later studies Me2SO was substituted by PVS2 (Leunufna and Keller, 2003; Panis et al., 2005). The droplet-vitrification method has benefited cryopreservation of yam (Leunufna and Keller, 2003), banana (Panis et al., 2005), Rosa (Halmagyi and Pinker, 2006), garlic (Kim et al., 2007) and taro (Sant et al., 2008).
Survival
Where effective, cold-hardening of donor plants tends to induce an intrinsic tolerance to low temperature and desiccation by triggering genes responsible for cold stress (Takagi, 2000). Tahtamouni and Shibli (1999) demonstrated that cold-hardening of the mother stock for 3 weeks at 4 °C under dark conditions followed by vitrification treatment improved survival and re-growth of cryopreserved shoot tips of wild pear. When the meristems of strawberry were cryopreserved by encapsulation-vitrification, those meristems cold-hardened at 4 °C for two weeks showed higher levels of shoot formation compared with non-hardened meristems (Hirai et al., 1998). Similar results were also observed in the present study (Table 2). Preculture of shoot tips with sucrose-enriched media prior to dehydration has been reported to be effective in improving post-thaw survival of some species, e.g. peach (Zhao and Wu, 2006). It is understood that the accumulation of sugars can slowly decrease the water content of material and increase the stability of membranes under conditions of severe dehydration (Crowe et al., 1989). For lily, the accumulation of sugars during pre-culturing on MS medium supplemented with 0.3 mol L−1 sucrose for 2 days was also confirmed to be very effective, as the present results showed (Fig. 3). The period during which the materials are treated with the dehydration/cryoprotective solution (PVS2) is also important, since it determines the extent of cell dehydration and the quantity of cryoprotectants permeating the cells. A long duration of PVS2 exposure was apparently toxic (Fig. 4). The optimal length of PVS2 treatment of different plant species varies considerably. For sweet potato shoot tips, exposure to PVS2 for 16 min after 60 min of loading treatment was optimal (Pennycooke and Towill, 2000), while for banana meristems, a high regeneration percentage was still obtained when the time of PVS2 treatment was 60 min (Panis et al., 2005). In this study, we found the optimal PVS2 treatment time for lily meristems was 90–120 min (Fig. 4).
Survival
Regeneration a
80
80
a
a
a
70
70 b
60 b
b
b
40
Percentage (%)
Percentage (%)
Regeneration
90
90
50
401
bc
30
c
a
60
a
50 40 30
b
b b
20
c
c
b
10
10 0
20
0
1
2
3
5
Preculture time (days) Fig. 3. Effect of time on medium with 30 g L−1 sucrose on the survival and regeneration percentages. Values are averages of three or four independent cryopreservation experiments. Error bars represent standard errors. Bars marked by the same letter are not significantly different for survival and regeneration percentages according to Duncan's Multiple Range Test. (P N 0.05).
0
0
60
90
120
150
PVS2 treatment time (min) Fig. 4. Effect of time exposed to PVS2 on the survival and regeneration percentages. Values are averages of three or four independent cryopreservation experiments. Error bars represent standard errors. Bars marked by the same letter are not significantly different for survival and regeneration percentages according to Duncan's Multiple Range Test. (P N 0.05).
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Table 3 Comparison of three lily cultivars cryopreserved by vitrification and droplet-vitrification. Cultivar
Survival and regeneration percentage
Droplet-vitrification
Vitrificat-ion method
Types of df and mean square
df
Mean square
F
Sig.
Lilium × siberia
Survival percentage (% ± S.E.)
65.0 ± 5.0
35.0 ± 3.5 35.0 ± 3.5
31.529
0.005
Survival percentage (% ± S.E.)
83.8 ± 8.5
45.3 ± 2.4
19.190
0.012
Regeneration percentage (% ± S.E.)
67.6 ± 9.1
36.3 ± 4.2
9.778
0.035
Survival percentage (% ± S.E.)
43.3 ± 6.7
41.7 ± 4.2
0.045
0.842
Regeneration percentage (% ± S.E.)
35.0 ± 2.5
33.3 ± 4.2
1350.000 55.830 1090.802 34.597 2227.227 116.062 1466.407 149.977 4.167 92.708 4.167 35.417
0.008
62.0 ± 3.3
1 4 1 4 1 4 1 4 1 4 1 4
24.181
Regeneration percentage (% ± S.E.)
Between groups Within groups Between groups Within groups Between groups Within groups Between groups Within groups Between groups Within groups Between groups Within groups
0.118
0.749
Lilium × Lancifolium
Lilium × Longiflorum
It is reported that the survival and regeneration percentages vary with different cultivars even using the same method (Leunufna and Keller, 2003; Panis et al., 2005). For example, among the 56 accessions of banana cryopreserved using the same method, the regeneration percentages varied from 19.8% to 84.7% (Panis et al., 2005). In the present study, the same cold-hardening, preculture, loading and the successive dropletvitrification procedures gave higher levels of shoot formation for two of the three lily cultivars tested (Table 3). Thus the preconditioning technique, such as cold-hardening, preculture, loading and the PVS2 treatment for enhancing dehydration or freezing tolerance appears variety-specific. In cryopreservation, it is of particular importance that the preserved meristems are capable of producing true-to-type plants identical to the non-treated phenotype. A callus phase prior to shoot formation is undesirable since callusing potentially increases the frequency of genetic variants (Haskins and Kartha, 1980). In this study, most cryopreseved shoot tips developed shoots directly without intermediary callus formation and grew to phenotypically normal plantlets (Fig. 1B–C). Regenerated plantlets could multiply when transferred to MS medium supplemented with 2 mg L−1 BA and 0.2 mg L−1 NAA, and produced normal bulblets with roots when placed on hormone-free MS medium. The morphological characteristics of plantlets developed from cryopreserved meristems such as plant height, leaf length, leaf width and leaf colour were observed and no morphological abnormalities were noted (Fig. 1D). Thus it appears that lily germplasm can be safely conserved by droplet-vitrification method.
5. Conclusion This study presents a protocol for cryopreservation of lily germplasm resources by droplet-vitrification, and compares the effects of droplet-vitrification and vitrification. We found droplet-vitrification to be preferable to vitrification. We believe that the droplet-vitrification method can benefit meristem cryopreservation of more plant species because of the higher cooling rate promoted. Finally, further study is necessary to
confirm the genetic stability of regenerated plantlets by cytological, biochemical and molecular analyses.
Acknowledgements This study was supported by National Key Technology R&D Program in accordance with the 11th five year plan of P. Rep. of China (No. 2006BA013B10), the special research grant of BasiC scientific Research Operation for Central Commonwealth Scientific Research Academy, the Ministry of Finance, P. Rep. of China (No.092060302-7) and the Crop Germplasm Resources Protection and Utilization Special Grant from the Ministry of Agriculture, People's Republic of China (No. NB08-2130135). Many thanks to Professor Johan Olausson (Institute of Botany, the Chinese Academy of Sciences) for his detailed revision and editing of this manuscript.
References Bouman, H., Tiekstra, A., Petutschnig, E., Homan, M., Schreurs, R., 2003. Cryopreservation of lilium species and cultivars. Acta Horticulturae 612, 147–154. Chen, H., Chen, X.L., Chen, L.Q., Lu, X.X., 2007. Cryopreservation of shoot tips from in vitro plants of cut flower of li1y (Lilium L.) by vitrification method. Journal of Plant Genetic Resources 2, 170–173 (in Chinese). Crowe, J.H., Crowe, L.M., Carpenter, J.F., Rudolph, A.S., Wistrom, C.A., Spargo, B.J., Anchordoguy, T.J., 1989. Interactions of sugars with membranes. Biochimica et Biophysica Acta 947, 367–384. Fabre, J., Dereuddre, J., 1990. Encapsulation-dehydration: a new approach to cryopreservation of Solanum shoot-tips. CryoLetters 11, 413–426. Fahy, G.M., Macfarlane, D.R., Angell, C.A., Meryman, H.T., 1984. Vitrification as an approach to cryopreservation. Cryobiology 21, 407–426. Fu, L.G., Chen, T.Q., Lang, K.Y., Hong, T., Lin, Q., Li, Y., 2002. Higher Plants of China. Qingdao Publishing House, Qingdao, pp. 118–133 (in Chinese). Halmagyi, A., Pinker, I., 2006. Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method. Plant Cell, Tissue and Organ Culture 84, 145–153. Haskins, R.H., Kartha, K.K., 1980. Freeze preservation of pea meristems: cell survival. Canadian Journal of Botany 58, 833–839. Hirai, D., Sakai, A., 1999. Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification. Plant Cell Reports 19, 150–155.
X.-L. Chen et al. / South African Journal of Botany 77 (2011) 397–403 Hirai, D., Shirai, K., Shirai, S., Sakai, A., 1998. Cryopreservation of in vitro grown meristems of strawberry (Fragaria ananassa Duch.) by encapsulationvitrification. Euphytica 101, 109–115. Kim, H.H., Lee, J.K., Hwang, H.S., Engelmann, Florent, 2007. Cryopreservation of garlic germplasm collections using the droplet-vitrification technique. CryoLetters 28, 471–482. Leunufna, S., Keller, E.R.J., 2003. Investigating a new cryopreservation protocol for yams (Dioscorea spp.). Plant Cell Reports 21, 1159–1166. Long, Y.Y., Zhang, J.Z., Zhang, L.N., 1999. Lily-corm Flower King. Jindun Press, Beijing. pp. 34–35. Matsumoto, T., Sakai, A., 1995. An approach to enhance dehydration tolerance of alginate-coated dried meristems cooled to −196 °C. CryoLetters 16, 299–306. Matsumoto, T., Sakai, A., Yamada, K., 1995. Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell, Tissue and Organ Culture 41, 237–241. Mix-Wagner, G., Conner, A.J., Cross, R.J., 2000. Survival and recovery of asparagus shoot tips after cryopreservation using the ‘droplet’ method. New Zealand Journal of Crop and Horticultural Science 28, 283–287. Panis, B., Piette, B., Swennen, R., 2005. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Science 168, 45–55. Pennycooke, J.C., Towill, L.E., 2000. Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification. Plant Cell Reports 19, 733–737. Sakai, A., 1995. Cryopreservation of germplasm of woody plants. In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry. : Cryopreservation of Plant Germplasm I, 32. Springer Berlin, pp. 53–69. Sakai, A., Kobayashi, S., Oiyama, I., 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9, 30–33.
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Sant, R., Panis, B., Taylor, M., Tyagi, A., 2008. Cryopreservation of shoot-tips by droplet vitrification applicable to all taro (Colocasia exculenta var. esculenta) accessions. Plant Cell, Tissue and Organ Culture 92, 107–111. Schäfer-Menuhr, A., Martin Schumacher, H., Mix-Wagner, G., 1997. Cryopreservation of potato cultivars: design of a method for routine application in genebanks. Acta Horticulturae 447, 477–482. Tahtamouni, R.W., Shibli, R.A., 1999. Preservation at low temperature and cryopreservation in wild pear (Pyrus syriaca). Advance in Horticultural Science 13, 156–160. Takagi, H., 2000. Recent developments in cryopreservation of shoot apices of tropical species. In: Engelmann, F., Takagi, H. (Eds.), Cryopreservation of Tropical Plant Germplasm. Current Research Progress and Application, IPGRI, Rome, Italy, pp. 178–193. Thinh, N.T., Takagi, H., Yashima, S., 1999. Cryopreservation of in vitro-grown shoot tips of banana (Musa spp.) by vitrification method. CryoLetters 20, 163–174. Towill, L.E., Bonnart, R., 2003. Cracking in a vitrification solution during cooling or warming does not affect growth of cryopreserved mint shoot tips. CryoLetters 24, 341–346. Uragami, A., Sakai, A., Nagai, M., 1990. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Reports 9, 328–331. Zhang, Y.Q., Li, X.X., Ma, Q., Wang, H.P., Shen, D., 2004. Preliminary study on vitrification cryopreservation technology of edible lily germplasm. China Vegetables 4, 11–13 (in Chinese). Zhao, Y.H., Wu, Y.Q., 2006. Cryopreservation of shoot tips from peach and its regeneration. Acta Horticulture Sinica 33, 1042–1044 (in Chinese).
South African Journal of Botany 77 (2011) 397 – 403 www.elsevier.com/locate/sajb
Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification X.-L. Chen, J.-H. Li, X. Xin, Z.-E. Zhang, P.-P. Xin, X.-X. Lu⁎ Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China Received 24 March 2010; received in revised form 9 October 2010; accepted 13 October 2010
Abstract In this study, apical meristems from adventitious buds of three lily (Lilium L.) cultivars were successfully cryopreserved by dropletvitrification. The most effective techniques were as follows. Excised meristems from in vitro plantlets which had been sub-cultured for about 2 months were cold-hardened at 4 °C for 1 week, precultured on MS medium supplemented with 0.3 mol L−1 sucrose for 2 days, osmoprotected in loading solution for 20–40 min at room temperature and then soaked in PVS2 solution for 90–120 min at 0 °C, frozen in microdroplets of vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen. The meristems were then rapidly rewarmed by dilution solution, transferred to regeneration medium and stored in the dark for two weeks at 20 °C, and then cultured under white fluorescent light at an intensity of 2000 lux, with a 16 h photoperiod at 20 °C. The highest post-thaw survival percentages of three cultivars ‘Siberia’ (Lilium × siberia), Lilium lancifolium Thunb. and ‘Snow Queen’ Lilium × longiflorum were 65.0%, 83.8% and 43.3%, and regeneration percentages were 62.0%, 67.6% and 35.0%, respectively. The study demonstrated that cryopreservation by droplet-vitrification increased survival and regeneration percentages of certain lily cultivars compared with vitrification. Thus to cryopreserve lily meristems, droplet-vitrification method is preferable to the vitrification method. Crown Copyright © 2010 Published by Elsevier B.V. on behalf of SAAB. All rights reserved. Keywords: Apical meristem; Cryopreservation; Droplet-vitrification method; Lily (Lilium L); Orthogonal experimental design
1. Introduction Lilies comprise a genus (Lilium L. in Liliaceae, Monocotyledoneae) of 115 species, of which 55 species originated in China. The genus is broadly distributed over China and species grow in different geographical areas, such as L. dauricum in Heilongjiang, L. martagon var. pilosiusculum in Xinjiang and L. formosanum in Taiwan (Fu et al., 2002; Long et al., 1999). Lilies are perennial bulbous herbs and since they are vegetative propagated, the germplasm resources cannot be preserved in low temperature seed banks. In vivo field preservation is labour intensive and there is a considerable risk of loss due to disease or extreme weather. On the other hand, in vitro tissue culture conservation is susceptible to contamination and somaclonal variation. Cryopreservation is the best choice for conservation
⁎ Corresponding author. Tel.: +86 10 62174099; fax: +86 10 62114309. E-mail addresses: [email protected], [email protected] (X.-X. Lu).
of lily germplasm resources due to its safety, repeatability and long-term storage possibilities. In 1973, the cultured cells of carrot were successfully cryopreserved for the first time. In the past 30 years, remarkable progress in cryopreservation has been made. Some simplified and valuable cryogenic techniques, such as the vitrification method (Sakai et al., 1990), air-drying method (Uragami et al., 1990), encapsulation-dehydration method (Fabre and Dereuddre, 1990) and the droplet-freezing method (Mix-Wagner et al., 2000; Schäfer-Menuhr et al., 1997) have been developed. Some new methods based on the combination of the last two methods have also been developed, such as encapsulation-vitrification method (Hirai and Sakai, 1999), and droplet-vitrification method (Leunufna and Keller, 2003). The number of species that can be cryopreserved has increased dramatically in recent years. For successful cryopreservation, it is essential to avoid lethal intracellular freezing which can occur during rapid cooling in liquid nitrogen (Sakai, 1995). Thus the materials to be cryopreserved have to be sufficiently dehydrated to avoid the
0254-6299/$ - see front matter. Crown Copyright © 2010 Published by Elsevier B.V. on behalf of SAAB. All rights reserved. doi:10.1016/j.sajb.2010.10.005
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lethal effects of intracellular ice crystals formation. To induce dehydration tolerance, materials need some treatments before being plunged into liquid nitrogen (LN), such as preculture on medium with a high sucrose concentration, treatment in loading solution, and exposure to plant vitrification solution (PVS). The injurious effects caused by the dehydration process are reduced by optimizing the duration of every step. The cooling rate is also a key factor for successful cryopreservation, because the ultra rapid freezing rate helps to avoid intracellular freezing and thus obtain a vitrified state during freezing (Fahy et al., 1984). Lilies have been cryopreserved by encapsulation-dehydration and vitrification in several previous studies (Bouman et al., 2003; Chen et al., 2007; Matsumoto and Sakai, 1995; Matsumoto et al., 1995; Zhang et al., 2004). Here we report for the first time the successful cryopreservation of meristems from adventitious buds of lily by droplet-vitrification. We also compared differences in survival percentage and regeneration percentage after cryopreservation between the droplet-vitrification and vitrification methods. Factors affecting the survival percentage and regeneration percentage were analyzed in order to provide a foundation for long-term and large-scale cryopreservation of lily meristems. 2. Materials and methods 2.1. Plant material The tissue culture of lily cultivar ‘Siberia’ (Lilium × siberia) was mainly used in the present study. Stock cultures of other two cultivars, Lilium lancifolium Thunb. and ‘Snow Queen’ (Lilium × longiflorum) were also used for comparison. The basal medium used for all the trials was Murashige & Skoog (MS) medium containing 3% sucrose and solidified with 7 g L−1 agar, and adjusted to pH 5.8 prior to autoclaving at 121 °C for 15 min. Adventitious buds were formed from the surface of bulb-scale segments after 30 days on MS medium with 6-benzylaminopurine (BA) 1.5 mg L −1 , α-Naphthalene acetic acid (NAA) 0.1 mg L−1, 250 mg L−1 casein hydrolysate, 3% sucrose, and 7 g L−1 agar. Then the adventitious buds were sub-cultured on MS medium with 2.0 mg L−1 BA, NAA 0.1 mg L−1, 3% sucrose, and 7 g L−1 agar to multiply under white fluorescent light (2000 lx) with a 16 h day−1 photoperiod at 20 °C for about 45 days. After sub-culture, they were grown on solidified MS medium in sterilized glass flasks under white fluorescent light at an intensity of 2000 lx with a 16 h photoperiod at 20 °C. 2.2. Cold-hardening and preculture In vitro plantlets sub-cultured for about 2 months were coldhardened at 4 °C for 1 week under white fluorescent light at an intensity of 2000 lx with a 16 h photoperiod. Meristems of about 1–2 mm in diameter with one or two leaf primordia were dissected from the cold-hardened and non-hardened segments under a binocular microscope in axenic conditions. The apical meristems were precultured in liquid MS basal medium supplemented with different sucrose concentrations (0, 0.2,
0.3, 0.4 and 0.5 mol L−1) for different days (0, 1, 2, 3 and 5 days) under the same light condition as indicated earlier. 2.3. Loading and vitrification procedure Pre-cultured meristems were osmoprotected in loading solution for 20–40 min at room temperature. The loading solution contained MS basal medium supplemented with 2 mol L−1 glycerol and 0.4 mol L−1 sucrose. The osmoprotected meristems were transferred onto sterilized filter paper for 30 s in order to absorb the exterior loading solution. The meristems were then soaked in PVS2 solution at 0 °C for various lengths of time (0, 60, 90, 120, and 150 min). The PVS2 solution consisted of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) dimethyl sulfoxide (Me2SO), and 0.4 mol L−1 sucrose in MS solution ( Sakai et al., 1990). In the droplet-vitrification treatment, 5–6 meristems were transferred to approximately 15-μl droplets of PVS2 solution on thin strips of sterile aluminium foil. The aluminium foil strips were folded to enclose the shoot tips. The folded foil strips were then carefully immersed into liquid nitrogen (LN) using fine forceps. After immersion, the strips were quickly transferred to 2 mL cryotubes which were immediately plunged into LN. In the vitrification treatment, meristems were transferred into 2 mL cryotubes containing 1 mL fresh ice-cold PVS2 and then plunged into LN. 2.4. Thawing and PVS2 dilution Samples were maintained in LN for at least 1 day. The meristems on the foil strips were then rapidly thawed by being immersed into 10 mL of dilution solution (DS) at room temperature for 15 min. DS consisted MS medium with 1.2 mol L−1 sucrose. The meristems were unloaded from the foil strips. This step also rinsed the PVS2 from the meristems. After 15 min, meristems were then immersed in 10 mL fresh DS for another 15 min. In the vitrification method, cryotubes were rapidly thawed in a water bath at 40 °C for 80 s. The PVS2 solution in the cryotubes was drained by a Pasteur pipette and replaced with DS. After 15 min, meristems were then kept for another 15 min in 10 mL fresh DS. 2.5. Plant recovery and viability After dilution of PVS2, meristems were placed onto regeneration medium composed of MS supplemented with 0.5 mg L−1 BA, 0.l mg L−1 NAA, 0.3 mg L−1 Gibberellic acid (GA3), 30 g L−1 sucrose, and 7 g L−1 agar and stored in the dark for two weeks at 20 °C. They were then cultured under white fluorescent light at an intensity of 2000 lx, with a 16 h photoperiod at 20 °C. The survival percentage was recorded as percentage of meristems that remained green after being cultured for one day in daylight. The regeneration percentage was recorded as percentage of the meristems producing normal shoots 4 weeks after planting.
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2.6. Experimental design
399
loaded for 20–40 min at room temperature and dehydrated with PVS2 for 90–120 min at 0 °C.
Orthogonal tests were applied for the study of dropletvitrification. Four factors and three levels of each factor were used and are listed in Table 1. Sucrose concentration in the preculture medium, duration of preculture and PVS2 treatment were analyzed by one-factor analysis in which one-factor was changeable and the other factors determined by the optimal treatment obtained from the orthogonal analysis results described in the Results section. Ten to twelve meristems were tested for each of the three repetitions per experiment.
2.7. Statistical evaluation
3.2. Responses of meristems after cryopreservation Four features distinguished meristems after plating on regeneration media for two weeks: (i) shoot tips becoming white indicated immediate tissue death after being plunged into LN; (ii) completely or partially black shoot tips indicated that there were reactions following cryopreservation (e.g. production and oxidation of polyphenols); (iii) callusing of meristems; and (iv) shoot tip regeneration (Fig. 1A1–4). 3.3. Cold-hardening
Results are presented as mean percentages with their standard error, and evaluated by SPSS 11.5 and Excel software. Significance of difference between mean values was assessed by analysis of variance (ANOVA) with Duncan's multiple range test (P b 0.05).
3. Results
The effects of cold-hardening on the survival percentage and regeneration percentage were summarized in Table 2. Post-thaw survival percentage and regeneration percentage of cold-hardened meristems were significantly higher than those of controls (P b 0.05), indicating the desirability of coldhardening. 3.4. Sucrose concentration during preculture
3.1. Orthogonal analysis The results of the orthogonal analysis are shown in Table 1. Except for the factor of loading time, results among all levels of the other three factors were significantly different. The order of significance was sucrose concentration N PVS2 treatment time duration of preculture N loading time. The optimal procedure led to the following recommendations: apical meristems should be cold-hardened for 1 week at 4 °C, precultured on MS medium supplemented with 0.3 mol L−1 sucrose for 2–3 days,
The sucrose concentration in preculture media significantly affected the post-thaw survival and regeneration percentages (Fig. 2), which increased at first, and then decreased with further increase in sucrose concentration. At 0.3 mol L−1 sucrose, survival and regeneration percentages peaked, reaching 69.2% and 63.8%, respectively. Adding more sucrose above 0.3 mol L−1 decreased the overall survival and regeneration capacity. 3.5. Preculture time
Table 1 Orthogonal design⁎ and analysis results. Treatment no.
Sucrose concentration (mol/L)
Preculture time (days)
Loading time (min)
PVS2 treatment time (min)
Survival percentage (%)
1 2 3 4 5 6 7 8 9 T1 T2 T3
0.3 (1) 0.3 (1) 0.3 (1) 0.4 (2) 0.4 (2) 0.4 (2) 0.5 (3) 0.5 (3) 0.5 (3) 51.9 a 21.5 b 16.9 b
1 (1) 2 (2) 3 (3) 1 (1) 2 (2) 3 (3) 1 (1) 2 (2) 3 (3) 18.5 b 31.2 ab 40.6 a
20 (1) 30 (2) 40 (3) 30 (2) 40 (3) 20 (1) 40 (3) 20 (1) 30 (2) 28.7 a 34.2 a 27.4 a
60 (1) 90 (2) 120 (3) 120 (3) 60 (1) 90 (2) 90 (2) 120 (3) 60 (1) 18.5 b 45.7 a 26.0 ab
27.3 72.7 55.6 10 8.3 46.2 18.2 12.5 20
The numbers in brackets refer to treatment level. T1–3 indicate the average value of the same level within the same column. Data within the same column followed by different letters are significantly different at the 0.05 level (Duncan's Multiple Range Test). ⁎ The orthogonal design is applied to preliminarily determine the optimal treatment as the basis of one-factor analysis in which one-factor was changeable and other factors were determined by the optimal treatment obtained from the orthogonal analysis.
Based on the sucrose concentration results, meristems were precultured in media with 0.3 mol L−1 sucrose for various lengths of time. Preculture time significantly affected the postthaw survival and regeneration percentages (Fig. 3) which were highest, 70.1% and 62.9%, respectively, when preculture was for 2 days. Percentages were significantly higher than for meristems precultured for 1 day or for the control. Post-thaw survival and regeneration percentages decreased steadily when preculture was for more than 2 days. 3.6. Time of PVS2 treatment Survival and regeneration percentages increased with increased exposure time to PVS2 and reached their highest when meristems were dehydrated for 90 min, then decreasing with further exposure (Fig. 4). However, the reduced percentages at 120 min were not significantly lower than those at 90 min. When dehydrated for 150 min, the survival percentage and regeneration percentage decreased to 15.3% and 7.4%, respectively. No meristem survived cryopreservation without PVS2 treatment.
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Fig. 1. Meristem responses after cryopreservation. Four responses of meristems after cryopreservation are illustrated. (A-1) whitened meristem; (A-2) blackened meristem; (A-3) callus; and (A-4) shoot development after cryopreservation. (B) Regenerated plantlets 4 weeks after cryopreservation; (C) regenerated plantlets 8 weeks after cryopreservation; and (D) regenerated plantlet after cryopreservation compared with control (untreated control to the left).
3.7. Comparison of the vitrification method and the droplet-vitrification method The three lily cultivars, ‘Siberia’, Lilium lancifolium Thunb. and ‘Snow Queen’ were cryopreserved by the droplet-vitrification and vitrification methods, and survival and regeneration percentages were compared. The results are shown in Table 3. ‘Siberia’ and L. lancifolium Thunb. responded significantly differently (P b 0.05) to the two methods. The survival and regeneration percentages when using droplet-vitrification method were significantly higher than those of the vitrification method. However, ‘Snow Queen’ meristems did not show any significant difference (P N 0.05) between the two methods. 4. Discussion In this paper, we compared two cryopreservation methods of droplet-vitrification and vitrification. Matsumoto and Sakai
(1995) and Matsumoto et al. (1995) cryopreserved lily meristems by encapsulation-dehydration and vitrification in previous studies and the percentage of shoot formation was high, but when we used their vitrification protocol the regeneration percentage was not as high as that achieved by those authors. The differences may have been caused by any of several factors, including donor plant variety, status, and culture conditions. For example, Panis et al. (2005) cryopreserved banana meristems using the protocol of Thinh et al. (1999), but the regeneration percentage was far lower than that achieved (by Thinh). However, in our study, for two of the three varieties tested, cryopreservation by droplet-vitrification relatively increased survival and regeneration percentages compared with vitrification. The greatest difference between the two protocols is freezing rate. The cooling rate in dropletvitrification which is about 130 °C s−1 is faster than that for vitrification of about 6 °C s−1 (Towill and Bonnart, 2003). Fahy et al. (1984) suggested that a high cooling rate helps obtain a
Table 2 Effects of cold-hardening on survival percentage and regeneration percentage. Control
Cold-hardened
Types of df and mean square
df
Mean square
F
Sig.
Survival percentage (% ± S.E.)
31.0 ± 4.6
63.1 ± 6.8 60.1 ± 5.1
1548.827 100.427 1660.007 56.117
0.017
26.8 ± 3.3
1 4 1 4
15.422
Regeneration percentage (% ± S.E.)
Between groups Within groups Between groups Within groups
29.581
0.006
Material: ‘Siberia.’
X.-L. Chen et al. / South African Journal of Botany 77 (2011) 397–403
Regeneration
Survival 90 a
80
a
Percentage (%)
70 60
b b
50 40
c
30 20
c
c
c c
c
10 0
0
0.2
0.3
0.4
0.5
Sucrose concentration (M) Fig. 2. Effects of preculture media with different sucrose concentrations on the survival and regeneration percentages. Values are averages of three or four independent cryopreservation experiments. Error bars represent standard errors. Bars marked by the same letter are not significantly different for survival and regeneration percentages according to Duncan's Multiple Range Test. (P N 0.05).
vitrified state during freezing. Droplet-vitrification developed from the simple droplet-freezing method, which was originally applied to shoot tips of potato (Schäfer-Menuhr et al., 1997) and asparagus (Mix-Wagner et al., 2000) when the cryoprotectant solution was 10% dimethyl sulphoxide (Me2SO/DMSO). In later studies Me2SO was substituted by PVS2 (Leunufna and Keller, 2003; Panis et al., 2005). The droplet-vitrification method has benefited cryopreservation of yam (Leunufna and Keller, 2003), banana (Panis et al., 2005), Rosa (Halmagyi and Pinker, 2006), garlic (Kim et al., 2007) and taro (Sant et al., 2008).
Survival
Where effective, cold-hardening of donor plants tends to induce an intrinsic tolerance to low temperature and desiccation by triggering genes responsible for cold stress (Takagi, 2000). Tahtamouni and Shibli (1999) demonstrated that cold-hardening of the mother stock for 3 weeks at 4 °C under dark conditions followed by vitrification treatment improved survival and re-growth of cryopreserved shoot tips of wild pear. When the meristems of strawberry were cryopreserved by encapsulation-vitrification, those meristems cold-hardened at 4 °C for two weeks showed higher levels of shoot formation compared with non-hardened meristems (Hirai et al., 1998). Similar results were also observed in the present study (Table 2). Preculture of shoot tips with sucrose-enriched media prior to dehydration has been reported to be effective in improving post-thaw survival of some species, e.g. peach (Zhao and Wu, 2006). It is understood that the accumulation of sugars can slowly decrease the water content of material and increase the stability of membranes under conditions of severe dehydration (Crowe et al., 1989). For lily, the accumulation of sugars during pre-culturing on MS medium supplemented with 0.3 mol L−1 sucrose for 2 days was also confirmed to be very effective, as the present results showed (Fig. 3). The period during which the materials are treated with the dehydration/cryoprotective solution (PVS2) is also important, since it determines the extent of cell dehydration and the quantity of cryoprotectants permeating the cells. A long duration of PVS2 exposure was apparently toxic (Fig. 4). The optimal length of PVS2 treatment of different plant species varies considerably. For sweet potato shoot tips, exposure to PVS2 for 16 min after 60 min of loading treatment was optimal (Pennycooke and Towill, 2000), while for banana meristems, a high regeneration percentage was still obtained when the time of PVS2 treatment was 60 min (Panis et al., 2005). In this study, we found the optimal PVS2 treatment time for lily meristems was 90–120 min (Fig. 4).
Survival
Regeneration a
80
80
a
a
a
70
70 b
60 b
b
b
40
Percentage (%)
Percentage (%)
Regeneration
90
90
50
401
bc
30
c
a
60
a
50 40 30
b
b b
20
c
c
b
10
10 0
20
0
1
2
3
5
Preculture time (days) Fig. 3. Effect of time on medium with 30 g L−1 sucrose on the survival and regeneration percentages. Values are averages of three or four independent cryopreservation experiments. Error bars represent standard errors. Bars marked by the same letter are not significantly different for survival and regeneration percentages according to Duncan's Multiple Range Test. (P N 0.05).
0
0
60
90
120
150
PVS2 treatment time (min) Fig. 4. Effect of time exposed to PVS2 on the survival and regeneration percentages. Values are averages of three or four independent cryopreservation experiments. Error bars represent standard errors. Bars marked by the same letter are not significantly different for survival and regeneration percentages according to Duncan's Multiple Range Test. (P N 0.05).
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Table 3 Comparison of three lily cultivars cryopreserved by vitrification and droplet-vitrification. Cultivar
Survival and regeneration percentage
Droplet-vitrification
Vitrificat-ion method
Types of df and mean square
df
Mean square
F
Sig.
Lilium × siberia
Survival percentage (% ± S.E.)
65.0 ± 5.0
35.0 ± 3.5 35.0 ± 3.5
31.529
0.005
Survival percentage (% ± S.E.)
83.8 ± 8.5
45.3 ± 2.4
19.190
0.012
Regeneration percentage (% ± S.E.)
67.6 ± 9.1
36.3 ± 4.2
9.778
0.035
Survival percentage (% ± S.E.)
43.3 ± 6.7
41.7 ± 4.2
0.045
0.842
Regeneration percentage (% ± S.E.)
35.0 ± 2.5
33.3 ± 4.2
1350.000 55.830 1090.802 34.597 2227.227 116.062 1466.407 149.977 4.167 92.708 4.167 35.417
0.008
62.0 ± 3.3
1 4 1 4 1 4 1 4 1 4 1 4
24.181
Regeneration percentage (% ± S.E.)
Between groups Within groups Between groups Within groups Between groups Within groups Between groups Within groups Between groups Within groups Between groups Within groups
0.118
0.749
Lilium × Lancifolium
Lilium × Longiflorum
It is reported that the survival and regeneration percentages vary with different cultivars even using the same method (Leunufna and Keller, 2003; Panis et al., 2005). For example, among the 56 accessions of banana cryopreserved using the same method, the regeneration percentages varied from 19.8% to 84.7% (Panis et al., 2005). In the present study, the same cold-hardening, preculture, loading and the successive dropletvitrification procedures gave higher levels of shoot formation for two of the three lily cultivars tested (Table 3). Thus the preconditioning technique, such as cold-hardening, preculture, loading and the PVS2 treatment for enhancing dehydration or freezing tolerance appears variety-specific. In cryopreservation, it is of particular importance that the preserved meristems are capable of producing true-to-type plants identical to the non-treated phenotype. A callus phase prior to shoot formation is undesirable since callusing potentially increases the frequency of genetic variants (Haskins and Kartha, 1980). In this study, most cryopreseved shoot tips developed shoots directly without intermediary callus formation and grew to phenotypically normal plantlets (Fig. 1B–C). Regenerated plantlets could multiply when transferred to MS medium supplemented with 2 mg L−1 BA and 0.2 mg L−1 NAA, and produced normal bulblets with roots when placed on hormone-free MS medium. The morphological characteristics of plantlets developed from cryopreserved meristems such as plant height, leaf length, leaf width and leaf colour were observed and no morphological abnormalities were noted (Fig. 1D). Thus it appears that lily germplasm can be safely conserved by droplet-vitrification method.
5. Conclusion This study presents a protocol for cryopreservation of lily germplasm resources by droplet-vitrification, and compares the effects of droplet-vitrification and vitrification. We found droplet-vitrification to be preferable to vitrification. We believe that the droplet-vitrification method can benefit meristem cryopreservation of more plant species because of the higher cooling rate promoted. Finally, further study is necessary to
confirm the genetic stability of regenerated plantlets by cytological, biochemical and molecular analyses.
Acknowledgements This study was supported by National Key Technology R&D Program in accordance with the 11th five year plan of P. Rep. of China (No. 2006BA013B10), the special research grant of BasiC scientific Research Operation for Central Commonwealth Scientific Research Academy, the Ministry of Finance, P. Rep. of China (No.092060302-7) and the Crop Germplasm Resources Protection and Utilization Special Grant from the Ministry of Agriculture, People's Republic of China (No. NB08-2130135). Many thanks to Professor Johan Olausson (Institute of Botany, the Chinese Academy of Sciences) for his detailed revision and editing of this manuscript.
References Bouman, H., Tiekstra, A., Petutschnig, E., Homan, M., Schreurs, R., 2003. Cryopreservation of lilium species and cultivars. Acta Horticulturae 612, 147–154. Chen, H., Chen, X.L., Chen, L.Q., Lu, X.X., 2007. Cryopreservation of shoot tips from in vitro plants of cut flower of li1y (Lilium L.) by vitrification method. Journal of Plant Genetic Resources 2, 170–173 (in Chinese). Crowe, J.H., Crowe, L.M., Carpenter, J.F., Rudolph, A.S., Wistrom, C.A., Spargo, B.J., Anchordoguy, T.J., 1989. Interactions of sugars with membranes. Biochimica et Biophysica Acta 947, 367–384. Fabre, J., Dereuddre, J., 1990. Encapsulation-dehydration: a new approach to cryopreservation of Solanum shoot-tips. CryoLetters 11, 413–426. Fahy, G.M., Macfarlane, D.R., Angell, C.A., Meryman, H.T., 1984. Vitrification as an approach to cryopreservation. Cryobiology 21, 407–426. Fu, L.G., Chen, T.Q., Lang, K.Y., Hong, T., Lin, Q., Li, Y., 2002. Higher Plants of China. Qingdao Publishing House, Qingdao, pp. 118–133 (in Chinese). Halmagyi, A., Pinker, I., 2006. Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method. Plant Cell, Tissue and Organ Culture 84, 145–153. Haskins, R.H., Kartha, K.K., 1980. Freeze preservation of pea meristems: cell survival. Canadian Journal of Botany 58, 833–839. Hirai, D., Sakai, A., 1999. Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification. Plant Cell Reports 19, 150–155.
X.-L. Chen et al. / South African Journal of Botany 77 (2011) 397–403 Hirai, D., Shirai, K., Shirai, S., Sakai, A., 1998. Cryopreservation of in vitro grown meristems of strawberry (Fragaria ananassa Duch.) by encapsulationvitrification. Euphytica 101, 109–115. Kim, H.H., Lee, J.K., Hwang, H.S., Engelmann, Florent, 2007. Cryopreservation of garlic germplasm collections using the droplet-vitrification technique. CryoLetters 28, 471–482. Leunufna, S., Keller, E.R.J., 2003. Investigating a new cryopreservation protocol for yams (Dioscorea spp.). Plant Cell Reports 21, 1159–1166. Long, Y.Y., Zhang, J.Z., Zhang, L.N., 1999. Lily-corm Flower King. Jindun Press, Beijing. pp. 34–35. Matsumoto, T., Sakai, A., 1995. An approach to enhance dehydration tolerance of alginate-coated dried meristems cooled to −196 °C. CryoLetters 16, 299–306. Matsumoto, T., Sakai, A., Yamada, K., 1995. Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell, Tissue and Organ Culture 41, 237–241. Mix-Wagner, G., Conner, A.J., Cross, R.J., 2000. Survival and recovery of asparagus shoot tips after cryopreservation using the ‘droplet’ method. New Zealand Journal of Crop and Horticultural Science 28, 283–287. Panis, B., Piette, B., Swennen, R., 2005. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Science 168, 45–55. Pennycooke, J.C., Towill, L.E., 2000. Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification. Plant Cell Reports 19, 733–737. Sakai, A., 1995. Cryopreservation of germplasm of woody plants. In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry. : Cryopreservation of Plant Germplasm I, 32. Springer Berlin, pp. 53–69. Sakai, A., Kobayashi, S., Oiyama, I., 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports 9, 30–33.
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Sant, R., Panis, B., Taylor, M., Tyagi, A., 2008. Cryopreservation of shoot-tips by droplet vitrification applicable to all taro (Colocasia exculenta var. esculenta) accessions. Plant Cell, Tissue and Organ Culture 92, 107–111. Schäfer-Menuhr, A., Martin Schumacher, H., Mix-Wagner, G., 1997. Cryopreservation of potato cultivars: design of a method for routine application in genebanks. Acta Horticulturae 447, 477–482. Tahtamouni, R.W., Shibli, R.A., 1999. Preservation at low temperature and cryopreservation in wild pear (Pyrus syriaca). Advance in Horticultural Science 13, 156–160. Takagi, H., 2000. Recent developments in cryopreservation of shoot apices of tropical species. In: Engelmann, F., Takagi, H. (Eds.), Cryopreservation of Tropical Plant Germplasm. Current Research Progress and Application, IPGRI, Rome, Italy, pp. 178–193. Thinh, N.T., Takagi, H., Yashima, S., 1999. Cryopreservation of in vitro-grown shoot tips of banana (Musa spp.) by vitrification method. CryoLetters 20, 163–174. Towill, L.E., Bonnart, R., 2003. Cracking in a vitrification solution during cooling or warming does not affect growth of cryopreserved mint shoot tips. CryoLetters 24, 341–346. Uragami, A., Sakai, A., Nagai, M., 1990. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Reports 9, 328–331. Zhang, Y.Q., Li, X.X., Ma, Q., Wang, H.P., Shen, D., 2004. Preliminary study on vitrification cryopreservation technology of edible lily germplasm. China Vegetables 4, 11–13 (in Chinese). Zhao, Y.H., Wu, Y.Q., 2006. Cryopreservation of shoot tips from peach and its regeneration. Acta Horticulture Sinica 33, 1042–1044 (in Chinese).