Introduction
Materials and Methods
Plant material
Cold hardening based on different tissues and preculture
Loading and dehydration procedure for cryopreservation
Unloading procedure and plant regrowth
Evaluation of survival and regrowth rates
Results and Discussion
Differences in cold-hardening according to tissues
Effects of loading solutions on survival and regrowth rates from cryopreserved bulblets
Effect of post-culture media on survival and regrowth rates from cryopreserved bulblets
Introduction
The genus Lilium is one of the most economically important plants of cut flowers in the horticulture and commercially grown as popular flowers in many countries. Maintaining the genetic diversity of these species is more demanding than for seed-producing plants, because it is difficult to get and maintain the disease-free sources (Reed, 2002). The genetic resources of Lilium could not be preserved long-term in low-temperature storages due to it is vegetative reproduction (Yi et al., 2013). Moreover, the attention and demand of consumer are rapidly changing about the flowering plants. Despite being an important genetic resource in the future, it quickly disappear the unpopular plants when new popular varieties are introduced to the commercial market. Also, the genus Lilium is facing the risks of serious loss or genetic erosion due to weak immune system caused by biological factors such as pathogens, diseases, pests or extreme climate and other factors.
Cryopreservation has been studied as a means of long-term preservation in various vegetative propagated plants such as sweet potato (Feng et al., 2011; Wang and Valkonen, 2008), potato (Dhital et al., 2009), and lily (Yi et al., 2013). In the previous studies for cryopreservation of various plants (Bouman and de Klerk, 1990; Bouman et al., 2003; Chen et al., 2011; Matsumoto et al., 1995; Yi et al., 2013; Yi et al., 2018), their results were obtained the high level of survival and regrowth after cryogenic temperature treatment. Thus, we tested to the efficiency of cold-hardening according to different tissues, such as only bulblets and bulb-scale with bulblets. The osmotic protection process is important to induce tolerance against dehydration and liquid nitrogen by using loading solution (LS) (Matsumoto et al. 1995; Sakai et al., 2008; Yamamoto et al., 2012). Studies on cryopreservation of various plants have shown that depending on the composition of the post-culture recovery media, which could affect the regeneration of the cryopreserved explants (Benelli et al., 2013; Li et al., 2015; Sant et al., 2008). The main purpose in the present study focused on to improve an efficient cryopreservation method by droplet-vitrification and to achieve high recovery rate from cryopreserved explants, that bulblets, such as adventitious buds, of Lilium spp.
Materials and Methods
Plant material
A lily, Oriental-Trumpet (OT) interspecific hybrid lily (Lilium spp.) cultivar named ‘Milky Way’, was used in this study for improvement of cryopreservation system in Lilium genetic resources. The lily accession was obtained from National Institute of Horticultural and Herbal Science (NIHHS) of the Rural Development Administration (RDA).
Cold hardening based on different tissues and preculture
For production of bulblets, which is adventitious buds, bulb scales was cultured on MS medium supplemented with 0.1 ㎎/L IAA, 0.1 ㎎/L zeatin, 30 g/L sucrose and 2.3 g/L phytagel for 2 weeks. We tested two types of cold hardening tissue parts, which the bulblets detached from bulb scale (Bulblet) (Fig. 1B) and the bulb scale with bulblets attached (Bulb-scale-bulblet) (Fig. 1A). First type, the bulblet collections was cold hardened on the solid MS medium included 0.3 M sucrose concentrations and second type, the bulb scale with bulblets on MS medium supplemented with 0.1 ㎎/L IAA, 0.1 ㎎/L zeatin, 30g/L sucrose and 2.3 g/L phytagel was cold hardened at 4℃ for 7 days in dark room. The bulblets attached with bulb scale were separated from the scale, prior to preculture. The collected bulblets were precultured in liquid MS medium containing 0.3 M sucrose for 31 hours and then, liquid MS medium containing 0.7 M sucrose for 17 hours in the dark room at 25℃. The bulblets of approximately 1-2 ㎜ in length were used for cryopreservation.
Loading and dehydration procedure for cryopreservation
The precultured bulblets were treated in loading solution (LS1, C4 or LS2, C6) containing 35% of PVS3 (LS1, C4) or 40% of PVS3 (LS2, C6) (Kim et al., 2009) for 40 min (Fig. 1C) and exposed to dehydration solution (PVS3, B1) (Kim et al., 2009) containing 50% glycerol and 50% sucrose for 60 min at 25℃ (Fig. 1D). The bulbs were moved onto droplets containing 3 µl PVS3 on sterilized aluminum foils (Fig. 1E), and then soaked into liquid nitrogen (LN) for at least 60 min (Fig. 1F).
Unloading procedure and plant regrowth
The bulblets on the foil strips in LN were rapidly immersed and thawed in 20 mL pre-heated unloading solution (40℃) which included liquid MS medium with 0.8 M sucrose (Fig. 1G), and then immediately removed half volumes of the unloading solution followed by another unloading solution (Room temperature, RT) added 10 mL volume at room temperature for 15 min. The bulblets were transferred to 10 mL fresh unloading solution (RT) for 25 min for cleaning of bulblets from dehydration solution. After these procedure steps, bulblets were cultured for survival and shoot regrowth in different post-cultured media (PCM). First recovery method, the bulblets cultured in MS medium without Ammonium nitrate (NH4NO3) including 3% sucrose, 1.0 g/L casein, 1.0 ㎎/L GA3, 0.5 ㎎/L BA (PCM1) for 5 days, and transferred to MS medium including 3% sucrose, 1.0 g/L casein (PCM2) for 9 days, followed by transferring to MS medium containing 3% sucrose, 15 ㎎/L putrescine, 0.2 ㎎/L zeatin, 0.15 ㎎/L IAA, and 0.05 ㎎/L GA3 (PCM3) for 6 weeks. Second recovery method, it cultured in PCM3 medium for 8 weeks.
Evaluation of survival and regrowth rates
The survival rate was surveyed the bulblets showing green shoot tips after 2 weeks of PCM medium, and then, the regrowth rate was evaluated after 8 weeks by counting the number of shooting plants that developed leaves and roots.
Results and Discussion
Previous the researches of cryopreservation were demonstrated that cold-hardening, osmoprotection, and post-culture medium among various cryopreservation steps were important as key role for increasing survival and regeneration of cryopreserved plants (Chen et al., 2011; Kim et al., 2012; Wang et al., 2003; Yamamoto et al., 2012). In this study, we examined three factors to find the efficient method of droplet-vitrification in Lilium spp. Briefly, cold hardening, loading solution, and post-culture media has different two levels, the levels of each factor are listed in Table 1.
Table 1.
The bulblets detached from bulb scale (Bulblet) collections was cold hardened on the solid MS medium included 0.3 M sucrose concentrations and the the bulb scale with bulblets attached (Bulb-scale-bulblet) on MS medium supplemented with 0.1 ㎎/L IAA, 0.1 ㎎/L zeatin, 30g/L sucrose and 2.3 g/L phytagel was cold hardened at 4℃ for 7 days in dark room. The bulblets were separated from the scale, prior to preculture. Precultured adventitious bulbs (MS + 0.3 M sucrose for 31 h and MS + 0.7 M sucrose for 17 h) were treated with loading solution containing 35% of PVS3 (LS1, C4) or 40% of PVS3 (LS2, C6) for 40 min and exposed to dehydration solution (B1) containing PVS3 (50% glycerol + 50% sucrose) for 90 min, prior to direct immersion in liquid nitrogen for 60 min. Cryopreserved adventitious bulbs were thawed in liquid MS medium with 0.8 M sucrose (40℃) for 10 sec and moved in liquid MS medium with 0.8 M sucrose (RT). Thawed adventitious bulbs were post-cultured in different post-cultured media (PCM) for 8 weeks.
Differences in cold-hardening according to tissues
The effects of cold-hardening on the survival and regrowth rates were showed in Table 2. We tested the effect of cold-hardening as the two tissues, which the bulblets detached from bulb scale (Bulblet) (Fig. 1B) and the bulb scale prior to excise bulblets from scale (Bulb-scale-bulblet) (Fig. 1A). The bulblets divided from bulb-scale-bulblet were used in preculture step. The post-cryopreservation survival and regrowth rates of the bulb-scale-bulblet and bulblet were showed as 68.6 and 65.7%, and 63.3 and 56.7%, respectively. The survival and regrowth rates bulb-scale-bulblet were higher than those of bulblets, indicating high efficiency of cold-hardening in bulb-scale-bulblet. Tahtamouni and Shibli (1999) reported that cold hardening of the mother plant improved the survival and regrowth rates of cryopreserved shoot tips in wild pear. We obtained the similar results in Lilium spp.
Table 2.
The bulblets detached from bulb scale (Bulblet) collections was cold hardened on the solid MS medium included 0.3 M sucrose concentrations and the the bulb scale with bulblets attached (Bulb-scale-bulblet) on MS medium supplemented with 0.1 ㎎/L IAA, 0.1 ㎎/L zeatin, 30g/L sucrose and 2.3 g/L phytagel was cold hardened at 4℃ for 7 days in dark room. The bulblets were separated from the scale, prior to preculture. Precultured adventitious bulbs (MS + 0.3 M sucrose for 31 h and MS + 0.7 M sucrose for 17 h) were treated with loading solution containing 35% of PVS3 (LS1, C4) for 40 min and exposed to dehydration solution (B1) containing PVS3 (50% glycerol + 50% sucrose) for 90 min, prior to direct immersion in liquid nitrogen for 60 min. Cryopreserved adventitious bulbs were thawed in liquid MS medium with 0.8 M sucrose (40℃) for 10 sec and moved in liquid MS medium with 0.8 M sucrose (RT). Thawed adventitious bulbs were post-cultured in MS medium containing 3% sucrose, 15 ㎎/L putrescine, 0.2 ㎎/L zeatin, 0.15 ㎎/L IAA, and 0.05 ㎎/L GA3 (PCM3 medium) for 8 weeks. Survival and Regrowth rate (%) were surveyed 2 and 8 weeks, respectively after post-culturing. The results are presented as means ± SD.
Effects of loading solutions on survival and regrowth rates from cryopreserved bulblets
Selection of suitable and stable loading solution (LS) is important for increasing efficiency of regrowth after treatment of LN, as it is highly sensitive to cytotoxicity (Benson et al., 1996; Rall and Fahy, 1985; Yamamoto et al., 2012). For selection of optimal LS, precultured bulblets were osmoprotected with two types of LS (Table 3). The survival and regrowth rates in control (-LN) without cryopreserve showed 83.8 and 80.6% for LS1 and 85.7 and 66.7% for LS2 in bulblets, and 88.1 and 84% for LS1 and 80.6 and 76.4% for LS2 in bulb-scale-bulblets. In cryopreserve (+LN), the results of them showed 63.3 and 56.7% for LS1 and 50 and 29.2% for LS2 in bulblets, and 68.6 and 65.7% for LS1 and 68.6 and 63.6% for LS2 in bulb-scale-bulblets as shown in Table 3. The regrowth rate was observed at a minimum level from cryopreserved (+LN) in bulblets, which treated with LS2. In these results showed that the survival and regrowth rates indicated higher in LS1 than LS2 in both bulblets and bulb-scale-bulblet. Therefore, loading solution (LS1) treatment is considered the cryoprotectant solution without or decreasing plant injury in Lilium spp. Mikyway. In bulblets, LS1 (C4-35%) was superior to LS2 (C6-40%), while in bulb-scale bulblets, both LSs produced similar post-cryopreservation survival and regrowth. It implies the possibility that bulblets (detached from the bulb scales) were stressed during the cold acclimation, compared to bulblets attached to bulb scales.
Table 3.
Precultured adventitious bulbs (MS + 0.3 M sucrose for 31 h and MS + 0.7 M sucrose for 17 h) were treated with loading solution containing 35% of PVS3 (LS1, C4) or 40% of PVS3 (LS2, C6) for 40 min and exposed to dehydration solution (B1) containing PVS3 (50% glycerol + 50% sucrose) for 90 min, prior to direct immersion in liquid nitrogen for 60 min. Cryopreserved adventitious bulbs were thawed in liquid MS medium with 0.8 M sucrose (40℃) for 10 sec and moved in liquid MS medium with 0.8 M sucrose (RT). Thawed adventitious bulbs were post-cultured in MS medium containing 3% sucrose, 15 ㎎/L putrescine, 0.2 ㎎/L zeatin, 0.15 ㎎/L IAA, and 0.05 ㎎/L GA3 (PCM3 medium) for 8 weeks. Survival and Regrowth rate (%) were surveyed 2 and 8 weeks, respectively after post-culturing. The results are presented as means ± SD.
Effect of post-culture media on survival and regrowth rates from cryopreserved bulblets
Various researchers in previous studies (Kim et al., 2012; Wang et al., 2003; Wang et al., 2017) reported that post-culture medium plays an important key role in the survival recovery of cryopreserved bulblets after cryopreservation in various plant species (Lambardi et al., 2000; Li et al., 2015; Sant et al., 2008). We investigated the efficiency of regrowth to check an optimal recovery medium by using three media, that the combination of the three media was used in two types (Tables 1 and 4). After cryopreserved steps, bulblets were cultured in types of different post-cultured media (PCM). As shown in Table 4, the survival and regrowth rates in control (-LN) without cryopreserve showed 80.9 and 76.2% for PCM1→PCM2→PCM3 media and 83.8 and 80.6% for PCM3 medium in bulblets, and 82.9 and 58.3% for PCM1→PCM2→PCM3 and 88.1 and 84% for PCM3 in bulb-scale-bulblets. In cryopreserve (+LN), the results showed 65.4 and 51.4% in PCM1→PCM2→PCM3 and 63.3 and 56.7% in PCM3, and 65.7 and 52.9% in PCM1→PCM2→PCM3 and 68.6 and 65.7% in PCM3 in the bulblets and bulb-scale-bulblets, respectively. After 8 weeks cultured on PCM media in cryopreserved (+LN), the highest regrowth rate showed that the better recovery as 65.7% of bulb-scale-bulblet cultured in only PCM3 medium. Based on the results in this study, consequently, It was decided that usages of cold-hardening bulb-scale-bulblet, loading solution (LS1), and recovery medium (PCM3) revealed that efficiency for increasing of cryopreservation. This result shows that processes of droplet-vitrification used in this study could be used as a promising method for long-term storage of lily genetic resource.
Table 4.
Precultured adventitious bulbs (MS + 0.3 M sucrose for 31 h and MS + 0.7 M sucrose for 17 h) were treated with loading solution containing 35% of PVS3 (LS1, C4) for 40 min and exposed to dehydration solution (B1) containing PVS3 (50% glycerol + 50% sucrose) for 90 min, prior to direct immersion in liquid nitrogen for 60 min. Cryopreserved adventitious bulbs were thawed in liquid MS medium with 0.8 M sucrose (40℃) for 10 sec and moved in liquid MS medium with 0.8 M sucrose (RT). Thawed adventitious bulbs were post-cultured in different post-cultured media (PCM). Survival and Regrowth rate (%) were surveyed 2 and 8 weeks, respectively after post-culturing. The results are presented as means ± SD.