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Cryopreservation of <I>Thymus Moroderi</I> by Droplet Vitrification

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Cryopreservation of <I>Thymus Moroderi</I> by Droplet Vitrification CryoLetters 31 (1), 14-23 (2010) © CryoLetters, [email protected] CRYOPRESERVATION OF Thymus moroderi BY DROPLET VITRIFICATION Ana Marco-Medina1, José Luis Casas1*, Rony Swennen2 and Bart Panis2 1Laboratory of Plant 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 plants 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.
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