CryoLetters 31 (1), 14-23 (2010) © CryoLetters, [email protected]

CRYOPRESERVATION OF moroderi BY DROPLET VITRIFICATION

Ana Marco-Medina1, José Luis Casas1*, Rony Swennen2 and Bart Panis2

1Laboratory of Biotechnology, Institute of Biodiversity CIBIO (Centro Iberoamericano de la Biodiversidad), University of Alicant, Crta. San Vicente del Raspeig s/n. E-03690 San Vicente del Raspeig, Alicante (Spain). 2Laboratory of Tropical Crop Improvement, Division of Plant Biotechnics, K.U. Leuven, Kasteelpark Arenberg 13, B-3001 Leuven, Belgium *Corresponding author e-mail: [email protected]

Abstract

Thymus moroderi Pau ex Martínez (Labiatae) was successfully cryopreserved using the droplet vitrification method. After 20 min in loading solution at room temperature, shoot tips were dehydrated with PVS2 at 0ºC for 30 min and immersed into LN. For thawing, shoot-tips were transferred into recovery solution for 15 min. A test of different recovery media revealed that the best results were obtained when the medium was supplement with 0.275 M BA. Keywords: cryopreservation, droplet vitrification, hyperhydration, in vitro conservation, Thymus moroderi

INTRODUCTION

Thyme species are well-known as medicinal, ornamental and aromatic and also used in pottery, cosmetics and perfumery. Many cultivars have been obtained as a result of cultivation (22). Thymus is widely distributed in the Old World. The Mediterranean region and more specifically the West Mediterranean area is considered as the centre of origin of this genus. Only species of two sections (sect. Sephyllum and sect. Hyphodromi subsect. Serpyllastrum) occur outside the Mediterranean area. Seven sections grow in the Iberian Peninsula and Northwestern Africa, five of them are endemic. In the Iberian Peninsula 35 species are found, 24 of which are endemic to that area (23). Interest in its use in folk medicine and its chemical components have resulted in uncontrolled harvesting, such that their populations are decreasing considerably. A continuously growing demand for thyme products is not likely to be supported by natural populations, which are threatened by destructive gathering and insufficient/irregular rainfall in traditional source areas (30). In vitro propagation methods for a limited number of species of Thymus have been already reported. These species are T. vulgaris (30), T. piperella (31) or T. mastichina (7, 20). In the present work we have focused on Thymus moroderi Pau ex Martinez, a species endemic to southeastern Spain characterised by deep purple bracts (23) and highly preferred in the

14 liqueur industry and folk medicine (1). This species is included in the Spanish red list of vascular plants (4) as near threatened according to the IUCN categories. In order to establish a cryopreservation protocol for this species, we decided to focus on the droplet vitrification method (26). This procedure is a combination of the droplet freezing method established by Schäfer-Menuhr et al. (36), and vitrification using the plant vitrification solutions designed by Sakai et al. (32). The main advantage of this technique compared to “normal” vitrification procedures is that it results in extremely high cooling and warming rates due to the very small volumes of cryoprotective medium in which the explants are placed (33). Droplet-vitrification is a very recent technique which until now has been applied to a few species such as papaya (2), Prunus (5), pelargonium (9), Chrysanthemum (11), yams (19), asparagus (21), banana (26), sweet potato (28) and taro (35). To our knowledge, this is the first report of cryopreservation of a member of Thymus species.

MATERIAL AND METHODS

Plant material All cryopreservation experiments were conducted with apical shoot tips excised from in vitro-grown plantlets of T. moroderi. To establish in vitro cultures, shoots collected from field-grown plants (El Pinaret, San Vicente del Raspeig, Alicante, Spain) were washed thoroughly with tap water and surface-sterilized by dipping in 500 mg l-1 benomyl 15 min, followed by 30 s in 70% ethanol and a further 15-min period in sodium hypochlorite 10% (v/v) solution. After rinsing 6 times with distilled and sterilized water in the laminar air flow cabinet, nodal segments were cultured on MS (24) medium with 30 g l-1 sucrose and solidified with 7 g l-1 plant agar. Medium pH was adjusted to 5.8 before autoclaving. Plantlets were grown at 25 ± 1°C, under an incident radiant flux of 40 mol m-2 s-1 and a 16 h-photoperiod. Explants from two clones differing in their age in vitro and in the number of subcultures were used. Clone 1 had been cultured in vitro for one year with a subculture every 6-8 weeks, while clone 2 was cultured for nearly 4 months under in vitro conditions.

Cryopreservation Cryopreservation was executed according to the droplet vitrification protocol developed for in vitro banana shoots (26). Excised 1-mm shoot tips were placed on solid MS medium (in a Petri dish wrapped with aluminium foil, to keep shoot tips in the dark) until all shoot tips were excised. Then, the shoot tips were placed in 30 ml plastic vials containing 3 ml of loading solution (LS: 0.4 M sucrose and 2 M glycerol in MS medium, pH adjusted to 5.8 followed by filter sterilization) (25) at room temperature. After 20 minutes the LS was removed with a Pasteur pipette and replaced by ice-cooled PVS2 (32). The vials were maintained on ice. Shoot tip dehydration with PVS2 was assayed for 0, 30, 60, 90, 120 and 180 min. The PVS2 solution consisted of 30% glycerol, 15% ethylene glycol, 15% DMSO and 0.4 M sucrose in MS medium, pH adjusted to 5.8 and filter sterilized. After PVS2 treatment, droplets (of about 15 l) of this solution containing shoot tips were placed onto sterile aluminium foil strips (0.5 x 2 cm in length) using a Pasteur pipette and then placed onto small Petri dishes on top of a cooling block (to keep the temperature of the strip around 0°C). Each aluminium strip containing 10 shoot tips was plunged (with the help of a sterile forceps) into a polystyrene box containing liquid nitrogen (LN). Samples remained in LN for at least 30 min.

15 Thawing For thawing, the aluminium foil strips were transferred with a sterile forceps to the recovery solution (1.2 M sucrose in MS medium, pH adjusted to 5.8 and filter sterilized) (32) at room temperature for 15 min. For this, the aluminium strips were stirred rapidly with a forceps in the recovery solution for rapid thawing thus releasing the shoot tips. After that, the shoot tips were transferred onto a sterile filter paper disc that was placed on top of a solid hormone-free MS medium containing 0.3 M sucrose and maintained in the dark for 24 h. Shoot tips were then transferred to regeneration medium (MS supplemented with 2.22 M BA) in which they were maintained one week in the dark before being transferred to light conditions (a 16 h light / 8 h dark photoperiod and a 50 µmol m-2 s-1 illumination provided by 36 W Osram cool-white fluorescent tubes.) Three replicates with ten shoot tips each were used for each PVS2 exposure time. Controls refer to replicates carried out in the same conditions as freezing experiments but without immersion in liquid nitrogen.

Recovery media For recovery, shoot tips were initially transferred to MS medium containing 2.22 M BA, 3% sucrose, 0.25% Gelrite™ and cultivated at 25 ± 2ºC in the light conditions indicated above. Due to a high incidence of hyperhydration in cryopreserved and recovering shoot-tips, different concentrations of BA (ranging from 0.275 to 2.22 M) and some combinations of BA with 0.25 M IBA (indol butyric acid) were tested. Additionally, 0.7% plant agar was used instead of 0.25% Gelrite™. During the optimization of the recovery medium, 30-min PVS2 dehydration was always applied. All experiments were repeated three times using 10 shoot tips for every medium. Three-four weeks after plating on MS supplemented with growth regulators, the surviving explants were transferred onto hormone-free MS medium for further outgrowth.

Assessment of survival and statistical analysis Survival was defined as shoot tips remaining green 2 weeks after thawing, while regrowth was defined as further development of apices into shoots 4 weeks after rewarming. Both survival and regrowth rates were expressed relative to the total number of shoot tips treated. Results are presented as mean percentages with standard error of mean (SE). Statistical differences between mean values of post-thaw survival and regeneration were assessed by analysis of variance (ANOVA) with Duncan’s multiple range test (P<0.05). The original percentage data were transformed by arcsin transformation (y’= arcsin y ½, y = original percentage/100). The analysis was made with SPSS 15.0 for Windows, SPSS. Inc., Chicago, IL.

RESULTS

Effect of length of PVS2 treatment without freezing In a first set of experiments, the toxicity of different exposure times of PVS2 (after a 20 min treatment with loading solution) towards large (2 mm) and small (0.5-1 mm) shoot tips of T. moroderi was evaluated without freezing. It has been generally accepted that an appropriate explant size for cryopreservation measures between 0.5 and 2 mm in length and consists of

16 the apical dome plus a couple of leaf primordia (39). It is observed that shoot tips of T. moroderi withstand relatively long dehydration times with PVS2 (Fig. 1). For the larger shoot-tips a reduction in survival was only obtained after 60 min treatment while smaller shoot tips appeared to be more sensitive. Here, a reduction in survival was already obtained after 15 min PVS2 treatment. Surprisingly, considerable survival was still obtained after 90 min of PVS2 exposure at 0°C and this for both shoot tip sizes.

100

a ab ab 80 ab ab A A

) 60 % (

l A A b a v i v

r A

u 40

S A

small 20 large

0 0 15 30 45 60 90 Exposure time (min)

Figure 1. Influence of PVS2 exposure on survival of small (1 mm) and large (2 mm) shoot- tips of T. moroderi. Survival was evaluated 2 weeks after excision. Vertical bars represent SE. Values followed by the same letter are not significantly different at the 0.05 probability level (Capital and normal letters should be considered separately). Values are the average of 4 independent experiments each containing 5-10 shoot tips.

Effect of length of PVS2 treatment with freezing In a subsequent set of experiments, we included a freezing step to test the cryoprotective role of PVS2. For this, explants were submitted to the same set of exposure times to PVS2 as described above and survival was evaluated 2 weeks after thawing. Although a slight improvement in the survival rate after PVS2 treatment was obtained using the larger shoot tips, we decided to conduct the rest of the cryopreservation experiments with the smaller ones to prevent a decay in survival after cooling to -196ºC, as was described by Takagi and coworkers (38).

100

80

a

) 60 % (

l ab a ab v i v r u

S 40 ab

bc 20

c 0 0 30 60 90 120 180 Exposure time (min) Figure 2. Effect of different exposure times to PVS2 at 0°C on 1-mm shoot tip survival after cryopreservation of T. moroderi. Survival was evaluated 2 weeks after thawing. Vertical bars represent SE. Values followed by the same letter are not significantly different (P<0.05). Values are the average of 3 independent experiments each containing at least 10 shoot tips.

17 One week after thawing, shoot-tips showed the first symptoms of survival: explants remained green, became a bit swollen and started to grow. A week later small shoots of 2-3 mm size developed. The highest survival rate was found after 30 min dehydration with PVS2 (Fig. 2). Statistical analyses (Duncan’s Multiple Range test) did, however, not reveal differences (p<0.05) with 60, 90 and 120 min of PVS2 dehydration. Survival started to decrease when dehydration was extended to 180 min (Fig. 2).

Effect of different recovery media after freezing Due to the high incidence of hyperhydration observed in those explants surviving cryopreservation (nearly 100% of surviving shoot tips showed this characteristic 2 weeks after thawing), shoot-tips were transferred to MS free-hormone medium to try to revert hyperhydricity. Hyperhydric shoots are named as such because they have a glassy appearance. Their stems and leaves are often thick, rigid and fragile (13). Many different parameters have been identified which can contribute to the occurrence of hyperhydricity, such as type of gelling agent and the presence of high amounts of cytokinins (10). Therefore, different concentrations of plant growth regulators were tested. Moreover, plant agar (7 g l-1) was added to the medium as gelling agent instead of Gelrite™ (2.5 g l-1). Seven different media were tested. As control, both MS free-hormone medium and MS supplemented with 2.22 M BA were employed. The latter was tested because it was used in previous experiments, in order to prove that the high incidence of hyperhydric shoots could be due to a high cytokinin concentration. Because the cytokinin BA is known to induce hyperhydricity when used at high concentrations (10), lower BA concentrations of 1.11, 0.55 and 0.275 M were also applied. Two weeks after thawing (Table 1), the medium supplemented with 1.11  or 0.275 M BA gave the highest survival rates (48.9% and 48.5% respectively), even though there were no significant differences with the other treatments, except with the medium supplemented with 1.11 M BA plus 0.25 M IBA (10.7%). Several types of morphogenetic responses could be distinguished 4 weeks after thawing (Table 1): 1. Good growth (regeneration):- explants developed into normal plantlets (Fig. 3a-b,e); 2. Slow growth:- explants were alive but grew slowly; 3. Hyperhydration:- explants showed the characteristic morphology of hyperhydricity, with black-brown coloured areas on their stem base (Fig 3c). After several weeks they definitively stopped growing making it impossible to revert the hyperhydration; 4. Growth with hyperhydration:- explants developed into slightly hyperhydric shoots but they still showed active growth. These explants should be transferred to hormone-free MS medium in order to attempt hyperhydration reversion (Fig 3d).

One month after thawing, shoot tips growing in medium supplemented with 0.275 M BA presented the highest regeneration rates (40%) and low level of hyperhydration (6.7%). Hyperhydric shoots were observed in all the treatments except in the basal medium without growth regulators, but shoot tips in this medium did not grow or grew very slow. As expected, higher hyperhydration rates (up to 26.7%) were observed when shoot tips were grown in the presence of the highest concentration of BA tested (2.22 M).

18 Table 1. Morphogenic response of cryopreserved T. moroderi shoot tips after thawing Morphogenic response

Hyperhyd- Growth with Survival Good growth ration Slow growth hyperhydration (%) (%) (%) (%) (%) MS 23.3 ± 8.8ab 0b 0b 13.3 ± 6.7a 0a 2.22 M BA 44.8 ± 14.4a 3.3 ± 3.3b 26.7 ± 8.8a 0b 13.3 ± 13.3a 1.11 M BA 48.9 ± 14.6a 20.0 ± 11.5ab 20ab 0b 13.3 ± 13.3a 0.55 M BA 20.0 ± 5.8ab 6.7 ± 3.3b 6.7 ± 3.3ab 0b 0a 0.275 M BA 48.5 ± 4.5a 40.0 ± 5.8a 6.7 ± 3.3ab 0b 0a 1.11 M BA + 0.25 M IBA 10.7 ± 0.4b 3.3 ± 3.3b 6.7 ± 3.3ab 0b 0a 0.275 M BA + 0.25 M IBA 28.9 ± 7.8ab 16.7 ± 8.8ab 3.3 ± 3.3b 0b 0a

Values are the average of 3 independent experiments each comprising 10 shoot tips. The difference between mean values in the same column was assessed by analysis of variance with Duncan’s multiple range test (P<0.05). Survival and morphogenetic response were evaluated 2 and 4 weeks after thawing, respectively.

Figure 3.A-B. Cryopreserved shoot tips growing in 0.275 M BA medium, three weeks after thawing. C. Hyperhydric shoots (in both tubes fasciated shoots can be observed) growing in MS medium solidified with Gelrite™ supplemented with 2.22 M BA. D. Cryopreserved explants one month after transfer to free-hormone MS medium (in the explant pointed out by the arrow, hyperhydration has been not overcome). E. Surviving explants from clone 1, two months after thawing. Bar = 1 cm.

19 Influence of age and number of subcultures on the explants’ response to cryopreservation In order to determine whether the response of explants to this protocol could be affected by the time spent in vitro and by the number of subcultures, we repeated the latter experiment using material that had been cultured in vitro for only 4 months with 3 subcultures. For this experiment, three replicates of 70 shoot tips were executed. Two weeks after thawing, high survival rates were observed in all medium tested (Table 2), survival ranged from 50.4% to 86.7%. Medium without hormones presented the lowest survival rates (50.4%) while media supplemented with cytokinins and auxins presented the highest rates (up to 86.7%).

Table 2. Effect of age and number of subcultures on the morphogenic response of T. moroderi explants

Morphogenic response

Hyperhyd- Growth with Survival Good growth ration Slow growth hyperhydration (%) (%) (%) (%) (%) MS 50.4 ± 26.2a 0c 0a 53.3 ± 2.0a 0c 2.22 M BA 86.7 ± 6.7a 26.7 ± 13.3b 0a 0b 63.3 ± 18.5a 1.11 M BA 79.2 ± 20.8a 0c 20.0 ± 20.0a 10.0 ± 10.0b 27.8 ± 14.7abc 0.55 M BA 71.7 ± 11.7a 25.0 ± 1b 0a 0b 43.3 ± 23.3ab 0.275 M BA 80.0 ± 15.3a 57.8 ± 13.1ab 0a 0b 16.7 ± 12.0bc 1.11 M BA + 0.25 M IBA 86.7 ± 6.7a 51.1 ± 16.4ab 10.0 ± 10.0a 10.0 ± 5.8b 20.0 ± 11.5abc 0.275 M BA + 0.25 M IBA 86.7 ± 8.8a 71.1 ± 10.6a 0a 0b 6.7 ± 6.7bc

Values are the average of 3 independent experiments each comprising 10 shoot tips. The difference between mean values in the same column was assessed by analysis of variance with Duncan’s multiple range test (P<0.05). Survival was evaluated 2 weeks after thawing while the morphogenetic response was evaluated 4 weeks after thawing.

Shoot tips were kept in these media for 4-6 weeks and the four parameters were evaluated. Medium without hormones presented only slow growth and medium with 2.22 M BA the highest level of hyherhydration. Media that presented the highest regeneration rates without hyperhydration were media supplemented with 0.275 M BA with or without auxins (71.1 and 57.8% respectively).

DISCUSSION

In this paper, an efficient cryopreservation method (droplet vitrification) is described for apical shoot tips excised from in vitro plantlets of Thymus moroderi Pau ex Martinez (Labiatae). To our knowledge, this is the first report on cryopreservation of a species of the genus Thymus. Cryopreservation protocols have already been developed for species from the genus and Mentha, also belonging to Labiatae. For example, cell suspensions of Lavandula vera have been successfully cryopreserved (40).

20 The genus Mentha has been more intensively studied. Towill reported the cryopreservation of mint shoot tips (Mentha aquatica x M. spicata) through vitrification (41): using a vitrification mixture that contains 35% ethylene glycol, 1 M dimethylsulfoxide and 10% polyethylene glycol-8000. Shoot tips from either in vitro or ex vitro mint plants have been cryopreserved by both two-step cooling and vitrification methods (42). In 1999, Hirai and Sakai (14) successfully cryopreserved alginate-coated meristems from in vitro grown axillary buds of Mentha spicata L. through vitrification. Towill and Bonnart (43) used in vitro plants of different mint species (Mentha aquatica, M. arvensis, M. piperita, M. Spicata ‘N83- 5’, M. spicata ‘Native’ and M. aquatica x M. spicata) to study the potentially damaging event that may occur during vitrification: i.e. cracking of the external glass formed by cooling and warming. Volk et al. (45) studied in shoot tips of Mentha x piperita L. potentially toxic combinations of cryoprotectant solutions; they exposed the material to one-, two-, three-, and four-component solutions of PVS2 chemicals at 0 and 22°C. Keller et al. (15) used a method which was a modified vitrification protocol based on the procedure published by Volk. Senula et al. (37) used droplet vitrification in Mentha x piperita L., M. x villosa and M. spicata L thus obtaining a recovery rate up to 89%. Recently, Uchendu and Reed (44) studied three cryopreservation protocols: controlled cooling, encapsulation-dehydration and vitrification in Mentha x piperita nothosubsp. citrata, M. canadensis, M. mistralis and M. cunninghamii. They obtained a regrowth rate of 93% using the controlled cooling protocol, followed by vitrification (73%) and encapsulation-dehydration (71%). The aim of this study was to develop a cryopreservation technique for Thymus moroderi that can be routinely applied in our laboratory for the ex situ conservation of this species. The method used is based on the droplet vitrification method established for banana meristems (26). This method has also been successfully applied to date palm embryogenic cultures (6), Pelargonium apices (9), potato apical shoot tips (27), olive embryogenic cultures (34), and taro shoot tips (35). In our study, the best results after cryopreservation were observed after 30 min PVS2 exposure, although survival was not significantly different from shoot tips treated for 60, 90 and 120 min according to Duncan’s Multiple Range test (p<0.05). A high resistance of thyme shoot tips towards PVS2 dehydration was observed. In other species, PVS2 treatment has been shown to be detrimental using short periods of incubation. In rose shoot tips (12) treatment with PVS2 for more than 20 min was damaging while in potato shoot tips (17) survival also decreased after 20 min of PVS2 treatment. Some authors found that modifications in the recovery medium could significantly improve the regeneration capacity of the cryopreserved explants of sweet potato (29), citrange (46) and kiwifruit (3). Especially variations in the growth regulator content of the recovery media influenced the percentage of regeneration. In garlic, however, no such influence could be observed (16). In a preliminary experiment with thyme shoot tips (data not shown), we observed that, after one week, shoot tips grew faster in medium supplemented with BA compared to a medium without growth regulators. However after a prolonged exposure to a high BA concentration shoot tips started to develop into hyperhydric shoots. This observation is similar to what was observed with grapevine (46). When cryopreserved shoot-tips were cultured on media with BA at concentrations higher than 1 M, a high frequency of hyperhydric shoots was obtained. Lambardi and co-workers (18) also observed symptoms of hyperhydricity in white poplar, particularly when explants were grown on a medium containing high concentrations of cytokinins. Similarly, Fukai and Oe (8) showed that the DMSO treatment during cryoprotection resulted in the development of hyperhydric shoots. In our study, survival and regeneration rates were higher using apical shoot tips belonging to clone 2 compared to clone 1. Both clones belonged to the same population, but

21 clone 1 was kept for more than one year in culture while clone 2 had been less than 4 months in culture. This probably causes differences in the morphological and physiological status of donor in vitro plantlets which can cause variations in post-thaw recovery (47). Finally, we can conclude that T. moroderi can be successfully cryopreserved using the droplet vitrification method. Acknowledgments: A.M.M. was financially supported by a Predoctoral fellowship (2004- 0640) and a travel grant from the Spanish Ministry of Science and Innovation. A.M.M. wish to thank the Laboratory of Tropical Crop Improvement (Leuven, Belgium) staff for their kindness and especially to Yves Lambeens for his assistance in the evaluation of the plant material.

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