Research Article

Korean Journal of Plant Resources. December 2020. 689-695
https://doi.org/10.7732/kjpr.2020.33.6.689

ABSTRACT


MAIN

  • 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.

/media/sites/kjpr/2020-033-06/N0820330616/images/kjpr_33_06_16_F1.jpg
Fig. 1

Cryopreservation of adventitious bulbs of lily (Lilium spp.) by droplet-vitrification. (A), bulb-scale culture for producing adventitous blubs for cold-hardening. (B), bulblet collections from bulb-scale for cold-hardening. (C), bulblets are treated with loading solution (LS1, C4) for 40 min at 25℃. (D), dehydrated with PVS3 solution (B1) for 90 min. (E), bulblets in droplets of PVS3 solution on sterilized aluminum foil strips. (F), Immersion of bulblets on the foil into the liquid nitrogen (LN) for 1 h. (G), unloading of cryopreserved bulblets on the foil strips in pre-heated solutions (40℃), MS+0.8M sucrose, for 40 min for thawing. (H), unloaded shoot tips on recovery culture medium and (I), plant regeneration from cryopreserved bulblets for 5 weeks after cryopreservation.

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.

Experiment procedures design for cryopreservation in Lilium spp.

Cultivar Cold hardening
(4℃)
Preculture Loading solution
(40 min)
Dehydration solution
(90 min)
Cryopreserved Post-culture media
Milky way Bulblet 0.3M
sucrose
(31hrs)
0.7M
sucrose
(17 hrs)
LS1 (C4) B1 -LN PCM1→PCM2→PCM3
+LN
-LN PCM3
+LN
LS2 (C6) -LN PCM1→PCM2→PCM3
+LN
-LN PCM3
+LN
Bulb-scale-bulblet LS1 (C4) -LN PCM1→PCM2→PCM3
+LN
-LN PCM3
+LN
LS2 (C6) -LN PCM1→PCM2→PCM3
+LN
-LN PCM3
+LN

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.

Effect of cold treatment on the survival and regrowth rate (%) of different tissues of the Lilium spp.

Cold-hardened tissues Cryopreserve Survival rate (%) Regrowth rate (%)
Bulblet -LN 83.8 ± 5.3 80.6 ± 0.9
+LN 63.3 ± 4.7 56.7 ± 4.7
Bulb-scale-bulblet -LN 88.1 ± 5.0 84.0 ± 0.9
+LN 68.6 ± 2.8 65.7 ± 1.4

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.

Effect of different loading solutions on the survival and regrowth rate (%) of cold-hardened different tissues before liquid nitrogen (-LN) and after (+LN)

Cold-hardened tissues Loading solution (40 min) Cryopreserve Survival rate (%) Regrowth rate (%)
Bulblet LS1 -LN 83.8 ± 5.3 80.6 ± 0.9
+LN 63.3 ± 4.7 56.7 ± 4.7
LS2 -LN 85.7 ± 20.2 66.7 ± 16.2
+LN 50.0 ± 11.8 29.2 ± 5.9
Bulb-scale-bulblet LS1 -LN 88.1 ± 5.0 84.0 ± 0.9
+LN 68.6 ± 2.8 65.7 ± 1.4
LS2 -LN 80.6 ± 3.9 76.4 ± 2.0
+LN 68.6 ± 16.2 63.6 ± 9.1

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.

Effect of different post-culture media on the recovery of cold-hardened different tissues after liquid nitrogen exposure (+LN)

Cold-hardened tissues Recovery media Cryopreserve Survival rate (%) Regrowth rate (%)
Bulblet PCM1→PCM2→PCM3 -LN 80.9 ± 1.3 76.2 ± 5.1
+LN 65.4 ± 17.6 51.4 ± 14.1
PCM3 -LN 83.8 ± 5.3 80.6 ± 0.9
+LN 63.3 ± 4.7 56.7 ± 4.7
Bulb-scale-bulblet PCM1→PCM2→PCM3 -LN 82.9 ± 4.0 58.3 ± 22.2
+LN 65.7 ± 8.1 52.9 ± 5.1
PCM3 -LN 88.1 ± 5.0 84.0 ± 0.9
+LN 68.6 ± 2.8 65.7 ± 1.4

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.

Acknowledgements

This study was carried out with the support of “Development and application of cryopreservation technique for strawberry and Lilium germplasm and quality management for seed base collection (Project No.PJ014294)”, National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea.

References

1
Benelli, C., A. De Carlo and F. Engelmann. 2013. Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Malus, Olea, Prunus, Pyrus and Vitis. Biotechnol Adv. 31:175-185. 10.1016/j.biotechadv.2012.09.00423022736
2
Benson, E.E., M. Wilkinson, A. Todd, U. Ekuere and J. Lyon. 1996. Developmental competence and ploidy stability in plants regenerated from cryopreserved potato shoot tips. Cryo Lett. 17:119-128.
3
Bouman, H. and G.J. de Klerk. 1990. Cryopreservation of lily meristem. Acta Hortic. 266:331-337. 10.17660/ActaHortic.1990.266.43
4
Bouman, H., A. Tiekstra, E. Petutschnig, M. Homan and R. Schreurs. 2003. Cryopreservation of Lilium species and cultivars. Acta Hortic. 612:147-154. 10.17660/ActaHortic.2003.612.18
5
Chen, X.L., J.H. Li, X. Xin, Z.E. Zhang, P.P. Xin and X.X. Lu. 2011. Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification. S. Afr. J. Bot. 77:397-403. 10.1016/j.sajb.2010.10.005
6
Dhital, S.P., H.T. Lim and H.K. Manandhar. 2009. Elimination of potato virus (PLRV and PVY) by cryopreservation of in vitro grown shoot tips of potato (Solanum tuberosum L.). Hort. Environ. Biotechnol. 50:233-239.
7
Feng, C.H., Z.F. Yin, Y.L. Ma, Z.B. Zhang, L. Chen, B. Wang, B.Q. Li, Y.S. Huang and Q.C. Wang. 2011. Cryopreservation of sweet potato (Ipomoea batatas) and its pathogen eradication by cryotherapy. Biotechnol. Adv. 29:84-93. 10.1016/j.biotechadv.2010.09.00220851757
8
Kim, H.H., E. Popova, D.J. Shin, J.Y. Yi, C.H. Kim, J.S. Lee, M.K. Yoon and F. Engelmann. 2012. Cryobanking of Korean allium germplasm collections: Results from a 10 year experiences. Cryo Lett. 33:45-57.
9
Kim, H.H., J.G. Lee, D.J. Shin, H.C. Ko, J.G. Gwag, E.G. Cho and F. Engelmann. 2009. Development of alternative plant vitrification solutions in droplet-vitrification procedures. Cryo Lett. 30:320-334.
10
Lambardi, M., A. Fabbri and A. Caccavale. 2000. Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips. Plant Cell Rep. 19:213-218. 10.1007/s00299005000130754897
11
Li, B.Q., C.H. Feng, M.R. Wang, L.Y. Hu, G. Volk and Q.C. Wang. 2015. Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet-vitrification and encapsulation-dehydration procedures. J. Biotechnol. 214:182-191. 10.1016/j.jbiotec.2015.09.03026432336
12
Matsumoto, T., A. Sakai and K. Yamada. 1995. Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell Tissue Organ. Cult. 41:237-241. 10.1007/BF00045087
13
Rall, W.F. and G.M. Fahy. 1985. Cryopreservation of mouse embryo at -196℃ by vitrification. Nature 313:573-575. 10.1038/313573a03969158
14
Reed, B.M. 2002. Implementing cryopreservation for long-term germplasm preservation in vegetatively propagated species. In Biotechnology in Agriculture and Forestry, Cryo- preservation of Plant Germplasm. 50:22-33. 10.1007/978-3-662-04674-6_2
15
Sakai, A., D. Hirai and T. Niino. 2008. Development of PVS-based vitrification and encapsulation-vitrification protocols: In Reed, B. (ed.), Plant Cryopreservation: A Practical Guide. New York: Springer LLC, USA. pp. 33-58. 10.1007/978-0-387-72276-4_3PMC2645499
16
Sant, R., B. Panis, M. Taylor and A. Tyagi. 2008. Cryopreservation of shoot-tips by droplet vitrification applicable to all taro (Colocasia esculenta var. esculenta) accessions. Plant Cell Tiss. Org. Cult. 92:107-111. 10.1007/s11240-007-9302-8
17
Tahtamouni, R.W. and R.A. Shibli. 1999. Preservation at low temperature and cryopreservation in wild pear (Pyrus syriaca). Adv. Hort. Sci. 13:156-160.
18
Wang, L.Y., Y.D. Li, H.Y. Sun, H.G. Liu, X.D. Tang, Q.C. Wang and Z.D. Zhang. 2017. An efficient droplet vitrification cryopreservation for valuable blueberry germplasm. Sci. Hortic. 219:60-69. 10.1016/j.scienta.2017.03.007
19
Wang, Q.C. and J.P.T. Valkonen. 2008. Efficient elimination of sweet potato little leaf phytoplasma from sweet potato by cryotherapy of shoot tips. Plant Pathol. 57: 338-347. 10.1111/j.1365-3059.2007.01710.x
20
Wang, Q.C., M. Mawassi, P. Li, R. Gafny, I. Selal and E. Tanne. 2003. Elimination of grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L. Plant Sci. 165:321-327. 10.1016/S0168-9452(03)00091-8
21
Yamamoto, S., K. Fukui, T. Rafique, N.I. Khan, M. Castillo, R. Carlos, K. Sekizawa, T. Matsumoto and T. Niino. 2012. Cryopreservation of in vitro-grown shoot tips of strawberry by the vitrification method using aluminum cryo-plates. Plant Gen. Res. 10:14-19. 10.1017/S1479262111000906
22
Yi, J.Y., G.A. Lee, J.W. Chung, S.Y. Lee and K.B. Lim. 2013. Efficient cryopreservation of Lilium spp. shoot tips using droplet-vitrification. Plant Breed. Biotech. 1(2):131-136. 10.9787/PBB.2013.1.2.131
23
Yi, J.Y., K. Balaraju, H.J. Baek, M.S. Yoon, H.H. Kim and Y.Y. Lee. 2018. Cryopreservation of Citrus limon (L.) Burm. F shoot tips using a droplet-vitrification method. Korean J. Plant Res. 31(6):684-694.
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