Agricultural and Food Science, vol. 18 (2009): 160-166 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 160–166. 160 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 160–166. 161 © Agricultural and Food Science Manuscript received April 2008 Research Note Application of vitrification-derived cryotechniques for long-term storage of poplar and aspen (Populus spp.) germplasm Ivaylo Tsvetkov1*, Carla Benelli2, Maurizio Capuana3, Anna De Carlo2, Maurizio Lambardi2 1 Forest Research Institute, 132, Kl. Ohridski Blvd., 1756 Sofia, Bulgaria *email: tsvet_i@yahoo.com 2 IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree, National Research Council (CNR), 50019 Sesto Fiorentino, Firenze, Italy 3 IGV/Istituto di Genetica Vegetale, National Research Council (CNR), 50019 Sesto Fiorentino, Firenze, Italy The application of three different vitrification-based freezing strategies for the cryostorage of white poplar (Populus alba L.) and hybrid aspen (P. tremula L. × P. tremuloides Michx.) have been as- sessed. The PVS2 vitrification protocol was successfully applied to two white poplar in vitro clones stored for more than 6 months in slow-growth conditions (4 °C, in darkness) and showing clear signs of explant etiolation and decay. After 60 min of PVS2 treatment, P. alba L. (cv. Villafranca) explants isolated from axillary buds demonstrated significantly better potential for post-freeze regrowth (64%) compared to those obtained from apical buds (17%). Similarly, a high level of survival (78%) of the frozen hybrid aspen shoot tips was recorded following the application of the same technique. Using the ‘encapsulation-vitrification’ procedure, no toxic effects of the PVS2 treatment were noticed after 120 min exposure, however none of the cryopreserved (poplar and aspen) explants survived after 3 weeks. In contrast, the ‘droplet-vitrification’ technique appeared to be very efficient in the cryopreservation of white poplar shoot tips, which increases the opportunities for wider application of this method in other woody species. Key-words: Aspen, cryopreservation, droplet vitrification, encapsulation-vitrification, Populus spp., PVS2 vitrification. A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 160–166. 160 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 160–166. 161 Introduction The poplar (Populus spp.) is rapidly being adopted as a model for studying different aspects of forest tree genetics for a number of reasons: its relatively small genome size, considerable genetic variation (both natural and resulting from breeding activity), fast juvenile growth, ease of in vitro clonal propagation and efficient transformation (Taylor 2002). Poplars are common throughout the northern hemisphere and well-adapted to a broad range of environments and climatic conditions. The white poplar (P. alba L.) is native to Central and Southern Europe and also found across Western Siberia and Central Asia in the riparian zones, as well as in natural forests. The species is appreciated for its ornamental value and broadly cultivated in urban areas. Among the white poplar cultivars in commercial use for years, the cv. Villafranca is that which has been intensively exploited in the production of timber and as a po- tential candidate for establishing ‘energy biomass’ plantations (Confalonieri et al. 2000). Although the poplar genome has recently been entirely sequenced (Tuscan et al. 2004), the genetic variability within the species would suggest that efforts to identify reliable methodologies for pre- serving its germplasm should be intensified. In this context, the cryogenic approach can be considered as a highly innovative strategy, offering unrivalled opportunities for cost-effective long-term germ- plasm preservation (Tsaiand Hubscher 2004). There have been successful attempts at apply- ing both slow-cooling (i.e. controlled-rate freez- ing) and ‘one-step freezing’ procedures for the cryopreservation of various poplar explants, such as twigs, buds, seeds and callus (Lambardi 2002). An efficient system for long-term cryopreservation of white poplar by vitrification was developed by Lambardi et al. (2000) and successfully applied, with some modification, also to the cryostorage of a hybrid (Populus tremula L. × Populus tremu- loides Michx.) aspen (Jokipii et al. 2004). How- ever, the step of experimental protocols tested on one or a few genotypes in a genus may produce variable results with different genotypes/cultivars (Reed 2001). Therefore, additional studies may be required to adapt established procedures so that they are applicable to a wider range of genotypes/ cultivars of interest. The present study aims to: test the opportunity to adapt and optimize • protocols based on PVS2 vitrification for the cryopreservation of white poplar (P. alba L.) in regard to other valuable Populus geno- types (clones, hybrids); evaluate the possibility of applying cryopro-• tocols to the preservation of shoot cultures, previously stored at 4°C for 6 months; develop and compare alternative one-step • cryotechniques (encapsulation-vitrification and droplet-vitrification). Materials and methods White poplar (P. alba L.) shoot cultures from cv. Vil- lafranca and from two clones (‘‘CSM’’ and ‘‘MLF’’) of significant interest in terms of germplasm preser- vation were used in this study. The two clones were established in vitro from ancient monumental white poplar trees located on the outskirts of Florence, Italy, representing local genetic resources.. Shoot cultures of these clones were maintained at 4 °C in darkness (slow-growth storage) for over 6 months and showed clear signs of etiolation and decay. In addition, material from a hybrid (P. tremula L. × P. tremuloides Michx.) aspen in vitro clone was also included, in a preliminary experiment. Three different techniques for cryopreservation (Fig. 1) were used. They were (A) the basic pro- cedure of cryopreservation by PVS2 vitrification (Lambardi et al. 2000) was tested with all the pop- lar and aspen clones/cultivars; (B) ‘encapsulation- vitrification’; and (C) ‘droplet-vitrification’. The last two techniques were applied to the ‘‘CSM’’ and ‘‘MLF’’ clones. The shoot cultures of both cv. Villafranca and the hybrid aspen were previously cold hardened for two weeks at 4 ºC under cool white fluorescent light (PPFD of 60 μmol m-2 s-1, 16 h photoperiod). Shoot tips (1.5–2 mm long) consisting of the api- A G R I C U L T U R A L A N D F O O D S C I E N C E Tsvetkov, I. et al. Application of cryotechniques for long-term storage of Populus spp. 162 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 160–166. 163 cal meristem and 4–5 leaflets were excised un- der a laminar flow hood from apical and axillary buds (cv. Villafranca), or from other apical buds (‘‘CSM’’, ‘‘MLF’’ and aspen). In all experiments, the excised shoot tips were precultured for 48 h on hormone-free MS medium (Murashige and Skoog 1962), under the same conditions described for cold hardening. The ‘CMS’ and ‘‘MLF’’ clones were used di- rectly for shoot-tip excision. Here, a pool of shoot tips were directly plated on MS medium without NH4NO3 and supplemented with 1.5 μM BA, 0.5 μM GA3 and 0.09M sucrose to test their potential to regrow when transferred to standard culture condi- tions (23 °C and 16 h photoperiod). Fig.1. Schematic representation of the different procedures applied for cryopreservation of poplar/aspen shoot tips: A) PVS2 vitrification; B) encapsulation-vitrification; and C) droplet-vitrification. A. PVS2 vitrification The shoot tips were loaded inside cryovials (2ml, 5–10 tips per cryovial) for 20 min at 25 °C with cryoprotectant solution (CP: 2M glycerol and 0.4M sucrose, Matsumoto et al. 1994). The cryoprotectant was then replaced with PVS2 solution (30% glycerol, 15% ethylene glycol and 15% DMSO, all w/v, in MS medium containing 0.4 M sucrose, Sakai et al. 1990). A 60-min exposure with PVS2 at 0 ºC was applied to the aspen and to both the ‘CSM’ and ‘MLF’ clones. Two different PVS2 exposure times (30 and 60 min) were tested with cv. Vil- lafranca shoot tips. After the PVS2 treatment, the shoot tips were suspended in 0.6 ml of fresh PVS2 and directly plunged into liquid nitrogen (LN) for at least one hour. A G R I C U L T U R A L A N D F O O D S C I E N C E Tsvetkov, I. et al. Application of cryotechniques for long-term storage of Populus spp. 162 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 160–166. 163 Fig. 2. Shoot tips of white poplar introduced into PSV2 drops on aluminium strips, placed on a frozen tile (left, bar = 5 mm) and then placed in 2.0 ml cryovials (right, bar = 5 mm). B. Encapsulation-vitrification For encapsulation, shoot tips were immersed in an MS liquid solution (lacking the calcium component), which contained 3% (w/v) Na-alginate. Using a pipette, the buds were then individually trans- ferred drop-by-drop (each drop containing a bud) to the complexing solution consisting of 100 mM CaCl2·2H2O and gently stirred to prevent the beads that had formed from sticking. The beads were then allowed to harden in the complexing solution for 20 min and retrieved using an autoclaved strainer. For cryopreservation, the beads were loaded with CP for 1 h at 25 °C, treated with PVS2 for 60 or 120 min at 0 °C, and finally plunged into LN. C. Droplet-vitrification With this procedure, droplets of PVS2 solution (10 μl) were put on small aluminium strips (0.5 × 2 cm), which were then placed on frozen tiles. One shoot tip was then introduced into each droplet and kept there for 30 and 60 min, respectively. Afterwards, the strips were placed in cryovials (containing LN) and then plunged in LN (Fig. 2). Rewarming and plating With each of the experiments, after at least 24 h storage at –196 °C, the shoot tips were rewarmed in a water bath at 40 ºC for either 1 min (PVS2 vitrification and droplet-vitrification) or 2 min (encapsulation-vitrification). The shoot tips (naked or encapsulated) were washed for 20 min at room temperature with liquid MS medium supplemented with 1.2 M sucrose, and were then plated on MS medium without NH4NO3 and supplemented with 1.5 μM BA, 0.5 μM GA3 and 0.09 M sucrose. Experimental design and statistical analysis The experimental design consisted of 20–25 explants per treatment and each experiment was repeated at least twice. According to the specific technique, 5-10 shoot tips or 5 beads or an aluminum strip with 3 explants, were placed in each cryovial. Frequency of survival was defined as a percentage (%) of green shoot tips that clearly demonstrate regrowth (i.e., showing leaf expansion/shoot development) three weeks after plating on regeneration medium. Data were subjected to chi-square (χ2) test for indepen- dence using SPSS statistical software (SPSS Inc.) A G R I C U L T U R A L A N D F O O D S C I E N C E Tsvetkov, I. et al. Application of cryotechniques for long-term storage of Populus spp. 164 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 160–166. 165 Results White poplar (Populus alba L.) After 6 months of storage at 4 °C in the dark, the shoot tips excised from the etiolated in vitro cultures of the ‘CSM’ and ‘MLF’ clones showed regrowth and the de novo-formed shoots resumed prolifera- tion in 2–3 weeks. When this material was used for the PVS2 vitrification trial, both ‘CSM’ and ‘MLF’ clones showed acceptable levels of survival, 54% and 44% respectively (Table 1). Following cryop- reservation, the shoot development was much faster in the clone ‘CSM’. The cv. Villafranca recorded a better survival of 64%, with shoot tips excised from axillary buds and treated with PVS2 solution for 60 min. This survival rate was significantly (p<0.001) higher than that of the apical buds (17%), as well as that of the shoot tips from both apical and axillary buds treated with PVS2 for 30 min (25% and 12% respectively). In our study, the ‘droplet-vitrification’ tech- nique improved the survival of shoot-tips, when compared to the PVS2 vitrification procedure. This effect was more evident with the clone ‘CSM’ where significantly (p<0.001) better survival (74%) of explants was observed following longer (60 min) PVS2 treatment (Table 2). To a lesser extent, the same effect was observed with the ‘MLF’ clone, but as distinct from the ‘CSM’ clone, peak survival of 55% was achieved after 30 min of PVS2 treat- ment. It is worth noting that, in general, the shoot tips took about 5 weeks on regeneration medium to convert into well-formed shoots. No post-freeze survival was achieved after the application of encapsulation-vitrification technique to both the white poplar ‘CSM’ and ‘MLF’ clones. The survival of the control shoot tips (encapsu- lated and unfrozen) was 67%, even after a 120-min PVS2 treatment (Fig. 3). White poplar clone/ cultivar Type of buds Survival % (–LN) Survival % (+LN) PVS2 30 min PVS2 60 min PVS2 30 min PVS2 60 min CSM Apical nt** 100.0 nt 53.8 MLF Apical nt 44.4 nt 44.2 ‘Villafranca’* Apical 100.0 a 100.0 a 25.0 a 16.7 a Axillary 100.0 a 100.0 a 12.0 a 64.3 b *Within the control (–LN) and liquid nitrogen (+LN) treatments, values followed by the same letter are not signifi- cantly different (p<0.001, Chi-square test). **nt – not tested Table 1. Survival of white poplar shoot tips 3 weeks after cryopreservation by ‘PVS2 vitrification’. White poplar clone Survival % (–LN) Survival % (+LN) PVS2 30 min PVS2 60 min PVS2 30 min PVS2 60 min CSM* 100.0 a 80.0 a 34.5 a 74.0 b MLF 88.9 a 100.0 a 54.5 a 44.0 a *For each clone and within the control (–LN) and liquid nitrogen (+LN) treatments, values followed by the same letter are not signif- icantly different (p<0.001, Chi-square test) Table 2. Survival of white poplar (P. alba L.) shoot tips 3 weeks after cryopreservation by ‘droplet-vitrification’ technique. A G R I C U L T U R A L A N D F O O D S C I E N C E Tsvetkov, I. et al. Application of cryotechniques for long-term storage of Populus spp. 164 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 160–166. 165 Hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) With the hybrid aspen, 78% survival was achieved after the shoot tips were treated with PVS2 for 60 min, directly immersed in LN, thawed and plated on regeneration medium (data not shown). Discussion The results from the cryopreservation of shoot tips excised from white poplar clones kept for longer than 6 months at ‘slow-growth storage’ revealed new opportunities for the direct cryostorage of this plant germplasm. Despite these explants showing clear symptoms of tissue decay prior to cryopreserva- tion, viable cultures regrew following exposure to LN. The successful cryopreservation results could possibly be explained by the acquisition of ‘cold hardiness’ during the ‘slow-growth storage’. In particular, the rates of survival obtained after PVS2 vitrification/one-step freezing open the door to future opportunities for the reliable use of this technique to cryopreserve plant material with altered physiology. In addition, the level of shoot tip survival from the preliminary study with the hybrid aspen was similar to that obtained by Jokipii et al. (2004) with the same species. These encouraging results obtained with the poplar clones demonstrate the potential of the PVS2 vitrification protocol to be directly used with other Populus genotypes of wider or unknown genetic origin. The results also indicate that the normal regrowth of non-frozen control explants after vitrification is not connected to possible PVS2 toxic effects, and so cannot explain the lack of survival following the ‘encapsulation-vitrification’ procedure. Hence, further experiments are required using longer exposure times to the vitrification solution of the beads. ‘Droplet-vitrification’ is a relatively new technique, up until now only applied to a restricted number of plant species (Sakai and Engelmann, 2007). Some recent reports however would seem to confirm that it is a promising tech- nique that will improve the tolerance of shoot tips to ultra-rapid freezing in LN (Panis et al. 2005, Sant et al. 2008). The results following the application of ‘droplet-vitrification’ are quite positive, and raise the possibility of further using this technique with other woody species. To the best of our knowledge, this is the first report describing the successful ap- plication of the ‘droplet-vitrification’ technique for cryostorage of forest species germplasm. Abbreviations: BA – benzyladenine, CP – cryoprotective solution, Fig. 3. Regrowth of white poplar encapsulated shoot tips (clone ‘MLF’) after PSV2 vitrification (non-frozen control). A) 120 min PVS2 treatment (bar = 10 mm); B) 60 min PVS2 treatment (bar = 10 mm). A G R I C U L T U R A L A N D F O O D S C I E N C E Tsvetkov, I. et al. Application of cryotechniques for long-term storage of Populus spp. 166 DMSO – dimethylsulfoxide, GA3 – gibberellic acid, LN – liquid nitrogen, MS – Murashige & Skoog, PPFD – photosynthetic photon flux density, PVS2 - plant vitrification solution 2 Acknowledgements. The study was done in Istituto per la Valorizzazione del Legno e delle Specie Arboree (IVALSA), Florence, during a 2-month grant (Short Term Scientific Mission) to I. Tsvetkov provided by COST Ac- tion 871 “Cryopreservation of Crop Species in Europe”. The technical assistance of Sara Pignatelli in helping with the experiments is greatly acknowledged. References Confalonieri, M., Belengi, B., Ballestrazzi, A., Negri, S., Facciotto, G., Schenone, G. & Delledonne, M. 2000. Transformation of elite poplar (Populus alba L. cv. Vil- lafranca) and evaluation of herbicide resistance. Plant Cell Reports 19: 978–982. Jokipii, S., Ryynanen, L., Kallio, P., Aronen, T. & Hagg- man, H. 2004. A cryopreservation method maintain- ing the genetic fidelity of a model forest tree, Populus tremula × Populus tremuloides Mincx. Plant Science (166) 3: 799–806. Lambardi, M., Fabbri, A. & Caccavale, A. 2000. Cryo- preservation of white poplar (Populus alba L,) by vitri- fication of in vitro-grown shoot tips. 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Application of vitrification-derived cryotechniques for long-term storage of poplar and aspen (Populus spp.) germplasm Introduction Materials and methods A. PVS2 vitrification B. Encapsulation-vitrification C. Droplet-vitrification Rewarming and plating Results White poplar (Populus alba L.) Hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) Discussion References