CRYOPRESERVATION OF IN VITRO-GROWN ...

CryoLetters 36 (3), 195- 204 (2015) © CryoLetters, [email protected]

CRYOPRESERVATION OF IN VITRO-GROWN SHOOT TIPS OF CHINESE MEDICINAL PLANT Atractylodes macrocephala KOIDZ. USING A DROPLET-VITRIFICATION METHOD Jin-mei Zhang1, Bin Huang2, Xin-xiong Lu1, Gayle M. Volk3, Xia Xin1, Guang-kun Yin1, Juan-juan He1 and Xiao-ling Chen1* 1

National Genebank, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 Fujian Agriculture and Forestry University, Fuzhou 350002, China; 3 USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521, U.S.A. *Corresponding author email: [email protected] Abstract BACKGROUND: Atractylodes macrocephala Koidz. is an important medicinal species from China that has been used for thousands of years for its special pharmacological antioxidant, hepatoprotective, anti-inflammatory, anti-allergic, antithrombotic, antiviral, and anticarcinogenic activities. OBJECTIVE: The aim of this research was to develop an efficient droplet-vitrification protocol for A. macrocephala shoot tips which could be used as a strategy for long-term conservation within gene banks. MATERIALS AND METHODS: The duration of preculture, loading, and PVS2 steps, as well as the recovery medium formulation, were optimized to achieve high levels of survival and regrowth for A. macrocephala shoot tips after liquid nitrogen exposure. RESULTS: Survival and regrowth levels after cryopreservation in the cultivar ‘Baizhu’ were as high as 76% and 62%, respectively. Thermal analysis using differential scanning calorimetry suggested that the PVS2 treatment plays a critical role for successful cryopreservation. CONCLUSION: The droplet-vitrification method established in this study could be used to cryopreserve A. macrocephala. Keywords: Atractylodes macrocephala Koidz., droplet-vitrification, regrowth, shoot tips, survival, thermal analysis pharmacological activities (7, 22, 39). Many components such as volatile oils, sesquiterpenoids, polysaccharides, amino acids, vitamins, resins and other ingredients have been identified in A. macrocephala (23, 51). Specifically, atractylol and atractylon are volatile oils that can be extracted from the roots. These oils have anti-inflammatory and anti-ulcer properties and they may inhibit lipid peroxidation and xanthine oxidase as well as the growth of esophageal carcinoma and tumor cells (52).

INTRODUCTION Atractylodes macrocephala Koidz., is a member of the Asteraceae family and has been used in traditional Chinese medicine for at least 2,000 years (51). The dried rhizome of A. macrocephala is an important ingredient of several Chinese herbal prescriptions because of its special antioxidant, hepatoprotective, antiinflammatory, anti-allergic, antithrombotic, antiviral and anticarcinogenic 195

Native populations of A. macrocephala have been exploited by collectors seeking plants for medicinal purposes and unauthorized trade. Its natural habitat is also at risk as a result of deforestation and urbanization. The plant has been domesticated and some cultivars are grown in Southern China in the Zhe Jiang, An Hui and Sichuan Provinces (25). A. macrocephala is highly outcrossing, and so propagation by seed may not produce progeny with the desired medicinal potential. Field propagation is time-, space- and labour-consuming and plants are susceptible to pathogen attacks and natural disasters. Thus, A. macrocephala germplasm is usually preserved in vitro (23, 24). Somaclonal variation and contamination are risks of tissue culture. Cryopreservation, the storage of biological materials at ultra-low temperatures in liquid nitrogen (LN, -196°C), while maintaining cell viability and the capacity to regenerate and grow after rewarming, is cost effective and space efficient (1). There are now more than 200 plant species that have been successfully cryopreserved (1, 9, 33). Medicinal plants, such as Dioscorea deltoidea (28), North American ginseng (Panax quinqueolius) (45), and Byrsonima intermedia (41) have been successfully cryopreserved, but a cryopreservation method for A. macrocephala has not yet been reported. The droplet vitrification technique, whereby shoot tips are placed in droplets of vitrification solution on foil strips and plunged into LN, has been successfully implemented to several crops, including yam, potato, taro, and lily (4, 8, 33, 37, 49). In this paper, we describe a straightforward procedure for cryopreserving A. macrocephala shoot tips, based on dropletvitrification. Prevention of intracellular ice formation is critical for successful cryopreservation. One of the challenges in developing cryopreservation methods is that there are many variables that must be optimized, including the subculture conditions, the 196

concentration of sucrose in preculture solution, the duration of preculture, loading and cryoprotectant exposures, and the recovery medium formulation (16, 18, 21, 38). Formation of ice and, sometimes, presence of glass formed by cryoprotective agents, can be detected in shoot tips, embryos and hairy roots using differential scanning calorimetry [(DSC); 11, 19, 42, 44, 47]. DSC measures the heat flow compared to a reference during tissue cooling and warming, and detects phase changes as well as water states (14, 47). In this study, we optimized four factors needed for the successful cryopreservation of A. macrocephala: preculture duration, loading duration, plant vitrification solution 2 [PVS2, 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) dimethyl sulfoxide] (36) exposure duration, and recovery medium formulations. Their effects on shoot tip survival and regrowth were investigated after LN exposure. Furthermore, DSC analysis was used to detect potentially deleterious ice in shoot tips at some critical steps of the cryopreservation procedure. MATERIALS AND METHODS Plant material In vitro propagated cultures of A. macrocephala Koidz. were maintained at the National Genebank of China, Institute of Crop Science, Chinese Academy of Agricultural Sciences (CAAS, Beijing, China). Two cultivars ‘Baizhu’ and ‘Anguo baizhu’ were used in this study. A. macrocephala plantlets were propagated by subculturing shoots monthly on Murashige and Skoog’s basal solid medium (31) supplemented with 2 mg L-1 benzyladenine (BA), 0.5 mg L-1 indole-3-butytric acid (IBA), 30 g L-1 sucrose and 8.0 g L-1 agar (pH 5.8) at 25 ± 2°C under 16 h light/8 h dark photoperiod (37.7 µmol m-2 s-1). To decrease the effects of the plant growth regulators on cryopreservation, the in vitro plantlets were transferred to hormone-free MS basal liquid medium for 7 to 15 days and then to hormone-free MS basal solid medium for 40 to 50 days.

days, loaded with 2.0 M glycerol and 0.4 M sucrose in MS basal medium at 25°C for 20 min, and then immersed in PVS2 at 0°C for 60 min. Shoot tips were then placed into PVS2 droplets on aluminum foil strips, plunged into LN, and then recovered on four recovery media (23, 25). These recovery media were: 1) MS basal solid medium supplemented with 30 g L-1 sucrose and 8.0 g L-1 agar; 2) MS basal solid medium with half strength of NH4+ and KNO3, 30 g L-1 sucrose and 8.0 g L-1 agar; 3) MS basal solid medium supplemented with 0.25 mg L-1 zeatin (ZT), 0.1 mg L-1 BA, 30 g L-1 sucrose and 8.0 g L-1 agar; and 4) MS basal solid medium supplemented with 0.3 mg L-1 BA, 0.02 mg L-1 1- NAA, 0.1 mg L-1 GA3, 30 g L-1 sucrose and 8.0 g L-1 agar.

Cryopreservation experiments In all experiments, the shoot tips (2-3 mm) of cultivar ‘Baizhu’ were excised from in vitro cultured plantlets and precultured in a liquid medium (MS basal medium with 0.3 M sucrose) in darkness on a shaker (SKY2102C, Sushen, Shanghai, China) at 25°C. The first experiment compared the length of the preculture duration (0, 1, 2, 3, 4 or 5 days), while keeping the subsequent steps unchanged: 20 min 2.0 M glycerol and 0.4 M sucrose in MS basal medium at 25°C, 60 min PVS2 at 0 °C, followed by transfer to PVS2 droplets on aluminum foil strips (5 x 30 mm). The loading step was optimized by performing an experiment whereby the preculture step was performed for 3 days, and then shoot tips were treated with 2.0 M glycerol and 0.4 M sucrose in MS basal medium at 25°C for 0, 10, 20, 30, 40 or 50 min. PVS2 exposure (0°C) was for 60 min, followed by transfer to PVS2 droplets on aluminum foil strips. The PVS2 step was optimized by preculturing for 3 days, loading in 2.0 M glycerol and 0.4 M sucrose in MS basal medium at 25°C for 20 min, and then immersing shoot tips in PVS2 at 0°C for 0, 20, 40, 60, 100 or 120 min, prior to transfer to PVS2 droplets on aluminum foil strips. Foil strips were directly plunged into cryovials (1.8 mL, Nunc, USA) filled with LN and kept in a LN tank (YDS-35-125, Dongya, Sichuan, China) in the liquid phase for 1 day. The shoot tips were rapidly rewarmed by dilution in 1.2 M sucrose unloading solution for 20 min. The shoot tips were transferred to solid recovery medium and cultured in the dark for 7 days at 25 ± 2°C, and then transferred to normal light intensity. For the above three experiments, the MS basal solid medium supplemented with 0.3 mg L-1 BA, 0.02 mg L-1 1-naphthaleneacetic acid (NAA), 0.1 mg L-1 gibberellin acid (GA3), 30 g L-1 sucrose and 8.0 g L-1 agar was used as recovery medium. A final experiment was performed using shoot tips that had been precultured for 3

Thermal analysis A Mettler-Toledo DSC 1 (MettlerToledo, Switzerland), connected to a liquid nitrogen cooling system and controlled with the STARe software package (version 6.00, Mettler-Toledo) was used to detect thermal transitions within ‘Baizhu’ shoot tips. Ten shoot tips (each 6.6 mg ± 0.6 mg, fresh weight) were weighed on a MettlerToledo microbalance (Mettler-Toledo, Switzerland), hermetically sealed into an aluminum pan. Samples were held at 0°C for 5 min, cooled to –150°C at 10°C per min and held at this temperature for 10 min, then warmed to 25°C at 10°C per min. Presence/absence of ice was measured in shoot tips that were: a) fresh (untreated) shoot tips; b) precultured for 1, 2, 3 or 4 days and then loaded with 2.0 M glycerol and 0.4 M sucrose for 20 min, followed by 60 min PVS2 at 0oC; c) precultured for 3 days, loaded with 2.0 M glycerol and 0.4 M sucrose for 0, 10, 20, 30, 40 or 50 min, followed by 60 min PVS2 at 0oC; and d) precultured for 3 days, loaded with 2.0 M glycerol and 0.4 M sucrose for 20 min, followed by 0oC PVS2 exposure for 20, 40, 60, 100 or 120 min. The onset of freezing was observed in DSC scans during cooling. Onset temperatures for melting were calculated from the intersection of the baseline and the 197

tangent to the steepest part of the peak, and the glass transition was calculated from the midpoint of the discontinuity in the baseline of the scan. Enthalpy of the ice melting was calculated from the area encompassed by the transition peaks and a projected linear baseline. Data analyses were carried out using STARe software. Thermograms from the DSC analysis were illustrated using GraphPad prism software (version 5.01, http//www. graphpad.com/).

Shoot tips were excised from subcultured plantlets of A. macrocephala cultivar ‘Baizhu’ (Fig. 1A, B) and some recovered plantlets were observed (Fig. 1C). A prolonged preculture appeared to have a slightly depressing effect on survival and regrowth levels after cryopreservation (Fig. 2A). Shoot tips precultured in 0.3 M sucrose in basal MS medium for 0 to 4 days had higher regrowth levels after LN exposure than those that were precultured for 5 days. Three day preculture treatments were performed in subsequent experiments. Shoot tips that were loaded for 20 min with 2.0 M glycerol and 0.4 M sucrose had significantly higher survival and regrowth than loading durations of 0, 10, 30, 40 or 50 min (Fig. 2B). The highest levels of survival and regrowth were obtained when PVS2 treatments were performed for 60 min (Fig. 3A). The composition of the recovery medium considerably affected survival and regrowth of shoot tips after cryoexposure (Fig. 3B). More than 60% survival and regrowth were obtained using MS solid medium supplemented with BA, NAA, and GA3 as the recovery medium. Considering the effects of these four factors on the cryopreservation, an optimized droplet-vitrification procedure for A. macrocephala ‘Baizhu’ shoot tips was established. Using the procedure optimized for cultivar ‘Baizhu’, the average survival and regrowth levels were 76 % and 62 %, respectively. For cultivar ‘Anguo baizhu’, the average survival and regrowth levels were 63% and 39 , respectively. In all the cryopreservation procedures

Data analysis Three to four treatment replications were performed for each experiment, and 10 to 20 shoot tips were tested for each replication. Survival of shoot tips was evaluated after 2 weeks by identifying shoot tips that remained green and had one or two leaves. The regrowth percentage was recorded as percentage of the shoot tips producing normal shoots after 4 to 6 weeks. Survival and regrowth were presented as the mean percentage with the standard deviation, and evaluated by SPSS 18.0 (http//www.spss.com/). Logarithmic transformations for survival and regrowth percentages were first executed before an analysis of variance (ANOVA) and the test of homogeneity of variances was performed. Least significant difference (LSD) or Dunnett T3 tests were performed to determine statistically significant differences among factors. RESULTS Development protocol

of

droplet-vitrification

Figure 1. A. In vitro plantlets, B. freshly excised shoot tips and C. regrowth from cryopreserved ‘Baizhu’ shoot tips, after one month. Note: Bar=1 mm in (B); Bar=1 cm in (C).

198

tested, shoots grew from cryopreserved shoot tips without intermediary callus formation. There were no morphological changes observed in the plantlets that were regenerated after cryopreservation.

tip dehydration and loading of cryoprotectants were initiated in the glycerol and sucrose step of the procedure. The size of the melting event changed dramatically because of the different treatments, with the largest peaks observed in fresh shoot tips (ca. 245.95 J g-1 fresh weight, Fig. 4A). The 20-min glycerol and sucrose loading treatment decreased the size of the endothermic peaks (ca. 120.74 J g-1 fresh weight) observed upon warming (Fig. 4A). After the 60 min PVS2 treatments, glass transition became apparent, and melting transitions were mostly not observed (Fig. 4A). ‘Baizhu’ shoot tips exposed to PVS2 solution for more than 20 min, regardless of the pretreatment or loading exposure duration, exhibited glass transitions during warming (Fig. 4B, 5A, 5B). The midpoint temperature of the glass transition remained

Thermal analysis Substantial water freezing and melting transitions were observed in DSC scans. Results showed that the temperature for the freezing onset decreased from about -14°C to about -19°C after exposure of shoot tips to sucrose preculture for 3 days, and significantly decreased to -51°C after exposure to glycerol and sucrose for 20 min. The onset temperature for melting decreased from about -1.19°C in the fresh controls and precultured shoot tips to -16.65°C after shoot tips were treated with glycerol and sucrose for 20 min (Fig. 4A). Treatment with glycerol and sucrose lowered the melting temperatures. It suggested that shoot

Figure 2. Effects of preculture duration (A) and loading duration (B) on survival and regrowth (%) of ‘Baizhu’ shoot tips after LN exposure. Note: Values are means of three replicate experiments. Error bars represent standard errors. Letters denote significant differences of the results for each treatment at the 0.05 level (LSD or Dunnett T3 test).

Figure 3. Effects of PVS2 exposure duration (A) and recovery medium (B) on survival and regrowth (%) of ‘Baizhu’ shoot tips after LN exposure. Note: Values are means of three replicate experiments. Error bars represent standard errors. Letters denote significant differences of the results for each treatment at the 0.05 level (LSD or Dunnett T3 test).

199

at -113°C regardless of the time in preculture, loading or PVS2 (Fig. 4B, 5A, 5B). In addition, recrystallization was sometimes observed as an exothermic event in warming DSC scans. In Fig. 4B, two small crystallization peaks were observed when shoot tips were precultured for 1 day,

about -40°C and 5°C, respectively. For the shoot tips which were precultured for 4 days, loaded for 20 min and treated with PVS2 for 60 min (Fig. 4B), one crystallization and one melting peak were observed. This result might be caused by crystallization and subsequent melting. In Fig. 5A, a melting peak was observed when shoot tips were

Figure 4. DSC thermograms of warming runs obtained using a Mettler-Toledo DSC1. ‘Baizhu’ shoot tips were processed through the cryoprotection protocol to the indicated step of the procedure and subsequently loaded into DSC: (A) freshly excised shoot tips, precultured for 3 days, loaded with 2 M glycerol and 0.4 M sucrose for 20 min, and PVS2 exposures of 60 min at 0 °C; (B) shoot tips were precultured for 1, 2, 3 or 4 days, loaded for 20 min, and then PVS2 for 60 min at 0 °C. Curves were vertically offset for better comparison. Gray arrows denote glass transition,white arrows denote freezing transitions and black arrows denote melting events.

Figure 5. DSC thermograms of warming runs obtained using a Mettler-Toledo DSC1. ‘Baizhu’ shoot tips were processed through the cryoprotection protocol to the indicated step of the procedure and subsequently loaded into DSC: (A) shoot tips were precultured for 3 days, loaded for 0, 20, 30, 40, or 50 min, and PVS2 for 60 min at 0 °C; (B) shoot tips were precultured for 3 days, loaded for 20 min, and PVS2 exposures of 20, 40, 60, 100 or 120 min at 0 °C. Curves were vertically offset for better comparison. Gray arrows denote glass transition,white arrows denote freezing transitions and black arrows denote melting events.

loaded for 20 min and treated with PVS2 for 60 min, and these two peaks crystallized at

precultured for 3 days, loaded for 10 min and treated with PVS2 for 60 min. For the 200

shoot tips which were precultured for 3 days, loaded for 40 min and treated with PVS2 for 60 min (Fig. 5A), some peaks were also observed. In Fig. 5B, a small peak was observed when shoot tips were precultured for 3 days, loaded for 20 min and treated with PVS2 for 20 min. DISCUSSION Droplet vitrification In recent years, cryopreservation has become a very important preservation method for the long-term storage of germplasm (9). Cryopreservation by dropletvitrification was shown in this study to be an effective method to safely preserve A. macrocephala in genebanks. In this study, the preculture duration affected the survival and regrowth levels of A. macrocephala shoot tips. The shoot tips precultured for 0, 1, 2, 3 or 4 days showed higher levels of regrowth after cryopreservation than those precultured for 5 days. Others have observed significant increases of sucrose concentrations in shoot tips during the preculture step (6, 46). This accumulation of sugar may slowly decrease the water content of materials and increase the stability of membranes (5). Sucrose may have multiple protective effects including non-lethal dehydration, membrane protection or cue to induce freezing tolerance metabolism (3). In this study, the loading and PVS2 treatment durations had significant effects on the survival and regrowth after cryopreservation. For many herbaceous species, the loading step after preculture in sucrose solution is very effective at inducing tolerance to freeze dehydration or cryoprotectant induced dehydration (33, 35). A. macrocephala had an optimal loading duration of 20 min, which is similar to that of many other species (29, 34, 48). PVS2 has high concentrations of penetrating cryoprotectants (DMSO and ethylene glycol) and osmotically active compounds (sucrose and glycerol) that reduce cellular water content and prevent ice 201

formation during cooling and rewarming of samples (10). Optimal exposure time to PVS2 varies by crop or cultivar (12, 20, 32). The length of the PVS2 exposure is very critical for successful cryopreservation, since it is important to find the correct balance between toxicity and adequate cellular dehydration to allow the cryoprotectant solution to enter cells without causing lethal damage (13). For A. macrocephala shoot tips, inadequate or excessive PVS2 exposure leads to the death of the meristem cells. We found the optimal duration of PVS2 treatment to be 60 min at 0°C. Recovery media were compared to identify a composition that resulted in healthy shoot tip recovery without callus. It was found that MS medium without activated charcoal and auxin helps the successful regeneration from cryopreserved quince (Cydonia oblonga) shoot tips (26). A medium with different concentrations of BA, NAA, GA3 and IAA stimulated production of fully grown plantlets of Dioscorea floribunda (7). In this study, the recovery medium significantly affected the regrowth level of cryopreserved A. macrocephala shoot tips. A. macrocephala shoot tips can not survive and regrow on MS without any plant growth regulators. The medium that included MS basal solid medium with half strength of NH4+ and KNO3 and the medium which included MS basal solid medium supplemented with 0.25 mg L-1 zeatin (ZT) and 0.1 mg L-1 BA, which are good for petunia and sweet potato in our lab (data not published), were not suitable for A. macrocephala survival and regrowth. The medium that included MS basal solid medium supplemented with 0.3 mg L-1 BA, 0.02 mg L-1 1- NAA and 0.1 mg L-1 GA3, based on the tissue culture medium in previous studies (21, 25), stimulated regrowth of ‘Baizhu’ healthy plantlets. BA and NAA are essential plant growth regulators for in vitro propagation and cryopreservation (17, 50). GA3 might enhance cellular proliferation and elongation (43). Another study found that successful

regrowth of shoot tips of Dioscorea alata required a 30 days culture duration on medium with BA, NAA and GA3 (30). Thermal behavior using dropletvitrification procedures DSC is used in fundamental cryobiology to guide the development of cryopreservation protocols. Profiling of crystallization and recrystallization events using DSC confirmed that freezing injury was minimized in samples after loading and cryoprotection with vitrification solution (40, 47). In this work, as expected, the size of melting event was reduced with PVS2 exposure and glass transitions became apparent. Loading and PVS2 reduced enthalpy, which is consistent with results published for other species (15, 17, 47). If glycerol, a common colligative additive, enters into the cells, it may increase the osmolality of the cell and reduce the efflux of water (1). PVS2 aids in the cryoprotection of shoot tips by replacing cellular water and changes the freezing behavior of the cellular water (47). ‘Baizhu’ shoot tips, treated with PVS2 for more than 20 min, showed minimal water freezing transitions in DSC experiments even though survival ranged from 0 to 70 %. This suggested that the absence of melting transitions after PVS2 exposure was not directly correlated with survival and regrowth. Significant glass transitions were observed after PVS2 exposure for 20 min, which suggested that this duration of PVS2 treatment sufficiently removed water and promoted vitrification. However, 60 min PVS2 was optimal for survival and regrowth, so additional cellular responses may occur during the longer duration that be necessary for higher levels of protection. Cryoprotection may need more time and subsequent molecular mobility and/or Hbonding properties of the system may be involved (2, 42). In conclusion, this study showed that droplet-vitrification could be successfully applied to A. macrocephala.

202

Acknowledgements: This study was supported by International Science & Technology Cooperation Program of China (2014DFG1860), Core Research Budget of the Non-profit Governmental Research Institution (ICS, CAAS, 2014JB02-002), the Crop Germplasm Resources Protection and Utilization Special Grant from the Ministry of Agriculture (2014NWB030-11), and the Agricultural Science and Technology Innovation Program (CAAS, ASTIP). REFERENCES 1. Benson EE (2008) Crit Rev Plant Sci 27, 141-219. 2. Buitink J & Leprince O (2004) Cryobiology 48, 215-228. 3. Carpentier SC, Vertommen A, Swennen R, Witters E, Fortes C, Souza MT & Panis B (2010) J Proteome Res 9, 50385046. 4. Chen XL, Li JH, Xin X, Zhang ZE, Xin PP & Lu XX (2011) S Afr J Bot 77, 397403. 5. Crowe JH, Crowe LM, Carpenter JF, Rudolph AS, Wistrom CA, Spargo BJ & Anchordoguy TJ (1988) Biochem Biophys Acta 947, 367-384. 6. Derreuddre J, Blandin S & Hassen N (1991) CryoLetters 12, 125-134. 7. Dong H, He L, Huang M & Dong Y (2008) Nat Prod Res 22, 1418-1427. 8. Dumet D, Diebiru E, Adeyemi A, Akinyemi O, Gueye B & Franco J (2013) CryoLetters 34, 107-118. 9. Engelmann F (2011) In Vitro Cell & Dev Biol Plant 47, 5-16. 10. Fahy GM, MacFarlane DR, Angell CA & Meryman HT (1984) Cryobiology 21, 407-426. 11. Gale S, Benson EE, Hardin & K (2013) CryoLetters 34, 30-39. 12. Halmagyi A, Delie C & Isac V (2010) Sci Hortic 124, 387-392. 13. Heringer AS, Steinmacher DA, Schmidt ÉC, Bouzon ZL & Guerra MP (2013) Protoplasma 250, 1185-1193. 14. Hohne GWH, Hemminger WF & Flammersheim HJ (2003) Differential

Scanning Calorimerty, Springer-Velag, Berlin Heidelberg. 15. Kaczmarczyk A, Funnekotter B, Turner S, Bunn E, Bryant G, Hunt TE & Mancera RL (2013) CryoLetters 34, 508-519. 16. Kaczmarczyk A, Shvachko N, Lupysheva Y, Hajirezaei MR & Keller ERJ (2008) Plant Cell Rep 27, 15511558. 17. Kalita V, Choudhury H, Kumaria S &Tandon P (2012) CryoLetters 33, 5868. 18. Kim HH, Lee YG, Park U, Lee SC, Baek JH, Cho EG & Engelmann F (2009) CryoLetters 30, 291-299. 19. Kim HH, Popova EV, Shin DJ, Bae CH, Baek HJ, Park SU & Engelmann F (2012) CryoLetters 33, 506-517. 20. Kim HH, Yoon JW, Park YE, Cho EG, Sohn JK, Kim TS & Engelmann F (2006) CryoLetters 27, 223-234. 21. Leunufna S & Keller ER (2005) CryoLetters 26, 93-102. 22. Li CQ, He LC, Dong HY& Jin JQ (2007) J Ethnopharmacol 114, 212-217 23. Li FL & Liu LQ (1992) J Wuhan Bot Res 3, 280-285 (In Chinese). 24. Liang XM, Zhu ZH & Hu XM (2009) J Anhui Agri Sci 37, 17356-17357 (in Chinese). 25. Liu XY, Wang L, Wang N, Li LH & Xu HD (2009) Chinese Wild Plant Res 28, 26-28 (in Chinese). 26. Lynch PT, Siddika A, Mehra A, Benelli C & Lambardi M (2014) CryoLetters 35, 188-196. 27. Mandal BB & Ahuja-Ghoshl S (2007) CryoLetters 28, 329-336. 28. Mandal BB & Dixit-Sharma S (2007) CryoLetters 28, 460-470. 29. Matsumoto T, Sakai A & Yamada K (1994) Plant Cell Rep 13, 442-446. 30. Mukherjee P, Mandal BB, Bhat KV, & Biswas AK (2009) CryoLetters 30, 100111. 31. Murashige T & Skoog F (1962) Physiol Plant 15, 473-497. 32. Panis B, Piette B & Swennen R (2005) Plant Sci 168, 45-55.

33. Reed BM (2008) Plant Cryopreservation: A Practical Guide. Springer, Berlin. 34. Reed BM, Uchendu EE, Muminova M & Gupta S (2010) In Vitro Cell & Dev Biol Plant 46, 386-393. 35. Sakai A & Engelmann F (2007) CryoLetters 28, 151-172. 36. Sakai A, Kobayashi S & Oiyama I (1990) Plant Cell Rep 9, 30-33. 37. Sant R, Panis B, Taylor M & Tyagi A (2008) Plant Cell Tissue Organ Cult 92, 107-111. 38. Sershen, Berjak P, Pammenter NW & Wesley-Smith J (2012) Protoplasma 249, 155-169. 39. Shan GS, Zhang LX, Zhao QM, Xiao HB, Zhuo RJ, Xu G, Jiang H, You, XM, & Jia TZ (2014) J Pharm Biomed Anal 98, 74-84. 40. Shin DJ, Kong H, Popova EV, Moon HK, Park SY, Park SU, Lee SC & Kim HH (2012) CryoLetters 33, 402-410. 41. Silva LC, Paiva R, Swennen R, Andre E & Panis B (2013) CryoLetters 34, 338348. 42. Sisunandar, Sopade PA, Samosir YMS, Rival A & Adkins SW (2010) Cryobiology 61, 289-296. 43. Soni M & Kaur R (2014) Physiol Mol Biol Plants 20, 95-101 44. Teixeira AS, Gonzalez-Benito ME & Molina-Garcia AD (2013) Biotechnol Prog 29, 707-717. 45. Uchendu EE, Brown DW& Saxena PK (2011) CryoLetters 32, 463-472. 46. Uragami A, Sakai A & Nagai M (1990) Plant Cell Rep 9, 328-331. 47. Volk GM & Walters C (2006) Cryobiology 52, 48–61. 48. Volk GM, Bonnart R, Krueger R & Lee R (2012) CryoLetters 33, 418-426. 49. Wang B, Wang RR, Li JW, Ma YL, Sheng WM, Li MF & Wang QC (2013) CryoLetters 34, 369-380. 50. Wang QC, Li P, Batuman O, Gafny R & Mawassi M(2003). CryoLetters 24, 293302.

203

51. Xu DN, Li WY, Huang YM, He JH & Tian YB (2014) Biol Trace Elem Res 160, 232-237. 52. Zhang JD, Cao G, Xia YH, Wen CP & Fan YS (2014) Pharmacog Mag 10, 249-253.

204