Impaginato 44 Adv. Hort. Sci., 2011 25(1): 44-50 Received for publication 24 February 2011. Accepted for publication 8 March 2011. Shoot-tip vitrification protocol for red chicory (Cichorium intybus L.) lines C. Benelli*, A. Previati**, A. De Carlo*, M. Lambardi* * Istituto per la Valorizzazione del Legno e delle Specie Arboree (IVALSA), Consiglio Nazionale delle Ricerche, Via Madonna del Piano, 10, 50019 Sesto Fiorentino (FI), Italy. ** Centro Sperimentale Orticolo “Po di Tramontana”, Veneto Agricoltura, Via Moceniga, 7, 45010 Rosolina (RO), Italy. Key words: cryopreservation, genetic resources, PVS2, RAPD, red chicory, vitrification. Abstract: Shoot tips from in vitro stock plants of red chicory ‘Rosso di Chioggia’ line were cryopreserved by one-step vitrification. After two days of cold-hardening on hormone-free MS medium and loading for 30 min in a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with PVS2 vitrification solu- tion at 0°C for 60 min and plunged directly into liquid nitrogen. The post-thaw survival of shoot tips was achieved 79% when was cultured on recovery medium containing 0.5 mM BA. Observed regrowth, after six weeks of culture in the same medium composition, was 100%. Rooted cryopreserved microshoots showed good quality when transferred to the greenhouse. Preliminary results proved that the genetic fidelity of the cryopre- served line was maintained. The same vitrification protocol was then applied to three other red chicory lines, ‘Rosso di Treviso precoce’, ‘Rosso di Treviso tardivo’ and ‘Castelfranco’. A simple and effective protocol for the cryopreservation of red chicory shoot tips has been successfully developed as a result of this study. 1. Introduction In Italy, the major production area of red chicory (Cichorium intybus L. var. intybus), covering about 9 thousand hectares, is located in the Veneto region, one of the most economically important areas for vegetable production. An ancient typology, ‘Rosso di Treviso Tardivo’, that was progenitor of the present varieties of red chicory, was introduced into Italy in the 15th cen- tury. Over time, growers selected those plants having good production, while in recent years this leafy veg- etable has undergone intense selection and breeding work, and several improved typologies have been pro- duced (such as ‘Rosso di Treviso precoce’, ‘Rosso di Verona’, ‘Rosso di Chioggia’, ‘Variegato di Castelfran- co’, and others) (Veneto Agricoltura, 2002), which are highly appreciated for their quality and productivity. At the “Po di Tramontana” experimental farm in Rosolina (Veneto Agricoltura, Rovigo, Italy) a specific breeding program to select high-performance lines has been continuing for many years. Every year after in- field evaluation, the most valuable lines are introduced and maintained in vitro by subculturing every three weeks. The stock plants obtained from these lines are transferred to the greenhouse to produce high quality seeds to be used for the production of high quality red chicory. The costs of stock culture maintenance and the risks of contamination and decay of lines can be reduced by introducing cryopreservation as a tool for a long-term preservation. Cryopreservation involves the maintenance of plant propagules at ultra-low tempera- tures (-196°C, LN): under these conditions, biochemi- cal and most physical processes are completely arrest- ed and as such, plant material can be stored for unlim- ited periods. Cryopreservation studies have been reported for Belgian endive (Cichorium intybus L. var. foliosum, cvs. Flash, Rumba and Carolus) by controlled-rate freezing (Demeulemeester et al., 1992; 1993) and encapsulation-dehydration techniques (Vandenbussche et al., 1993). Controlled-rate freezing is regarded as the traditional approach to plant cryopreservation. Although controlled-rate freezing has been effective for cryopreserving differentiated tissues (Reed and Uchendu, 2008), reports on cryostorage using this tech- nique are limited. One of the practical limitations of the controlled-rate freezing approach is the need for an expensive programmable freezer (Engelmann, 2000). However, applying traditional (controlled-rate freez- ing) and new procedures (one-step freezing technique), the first examples of “cryogenic banks” are available today in several countries (Reed, 2001; Sakai and Engelmann, 2007). At the same time, the development 45 of a vitrification system has made it possible to increase the number of plant species that can be cryop- reserved (Towill and Bajaj, 2002; Lambardi and De Carlo, 2003, 2009; Sakai et al., 2008). In the present study, a vitrification/one-step freez- ing procedure was developed using ‘Rosso di Chiog- gia’ shoot tips, with exposure of explants to the vitrifi- cation solution and rapid cooling by directly immersing in liquid nitrogen (LN). This procedure was then applied to three different red chicory typologies. In addition, validation of this method using molecular markers is presented with regard to maintenance of genetic stability. 2. Materials and Methods Plant material Microshoots of red chicory ‘Rosso di Chioggia (mp C7 4/212) were adventitiously induced from leaf por- tions through direct organogenesis (Fig. 1 a). The lines were obtained by isolating each adventitious shoot and transferring them onto a semi-solid MS (Murashige and Skoog, 1962) medium, supplemented with 30 g l-1 sucrose, 1.0 µM 6-benzyladenine (BA) and 7 g l-1 agar (proliferation medium). The pH of the medium was adjusted to 5.8 before autoclaving for 20 min at 121°C. The shoot cultures were placed at 21±1°C under Fig. 1 - Cryopreservation of red chicory “Rosso di Chioggia” line (a-f). a - microshoots induced adventitiously from a leaf portion; b - a shoot tip just after excision from a shoot bud; c - shoot tip survival after three weeks and a well-formed shoot tip after cryopreservation with 60 min of PVS2 treatment (detail); d - regrowth in plastic cylinder on MS medium with 0.5 µM BA; e - cryopreserved rooted microshoot; f - plantlets from cryopreservation, after potting and acclimation in the greenhouse. 46 12-hr photoperiod conditions (60 µmol m-2 s-1 photo- synthetically active radiation) and subcultured every three weeks (standard culture conditions). Short-day conditions avoided floral initiation. Vitrification procedure Before bud excision, stock plants were exposed to 4°C with an 8-hr photoperiod for a three-week cold- hardening period. Shoot tips (2-3 mm long), consist- ing of the apical meristem and 4-5 leaflets, were then excised from microshoots (Fig. 1b) under a stereo microscope in aseptic conditions, and were precul- tured on hormone-free MS medium supplemented with 0.09, 0.3 or 0.7 M sucrose for two days at 4°C under an 8-hr photoperiod to determine the effect of sucrose pretreatment on cryopreserved explant sur- vival. To induce dehydration, the shoot tips were loaded with a cryoprotectant (Loading Solution, LS; 2 M glycerol and 0.4 M sucrose) (Matsumoto et al., 1994) for 30 min at 25°C in 2-ml Nalgene® cryovials (10 shoot-tips per cryovial) and subsequently incubated at 0°C with the PVS2 (Plant Vitrification Solution 2; 30% w/v glycerol, 15% w/v ethylene glycol, 15% w/v DMSO in MS medium containing 0.4 M sucrose) (Sakai et al., 1990). In order to choose the longest possible exposure time of shoot tips to the vitrifica- tion solution while avoiding toxic effects, shoot tips were exposed to PVS2 for 30, 60, 90 or 120 min and evaluated for shoot tip survival. After dehydration, the samples were suspended in 0.6 ml of fresh PVS2 solution and rapidly frozen to –196°C by direct immersion in LN, where they were stored for at least 2 hr. For recovery, they were quickly rewarmed by plunging the cryovials into a 40°C waterbath (warm- ing rate: about 150°C min-1), unloaded from the PVS2 solution, washed for 20 min at 25°C in a liquid MS medium containing 1.2 M sucrose and finally plated onto the proliferation medium and maintained under standard culture conditions. Cryopreserved shoot tips were assessed for survival after three weeks. In addition to proliferation medium, five different recovery media were tested to improve the regrowth of cryopreserved shoot tips: MS added with 0.5, 1.0 or 5.0 µM BA, MS with 1 µM Thidiazuron (TDZ) and MS with 0.5 g l-1 Activated Charcoal (AC). The role of AC has been discussed in many reports on plant tis- sue culture with different effects (Thomas, 2008). Survival was defined as a percentage of green shoot tips after three weeks from thawing. Explants were transferred to plastic tubes (one shoot for each tube) and six weeks after LN treatment the percentage of plants demonstrating regrowth was assessed. Explants treated with PVS2 but not exposed to LN were used as controls. In vitro rooting of cryopreserved shoots Cryopreserved shoots were transferred to jars con- taining 100 ml of MS rooting medium with 20 g l-1 sucrose, 7 g l-1 agar and supplemented with 2.5 µM indole butyric acid (IBA). The jars were kept in a cli- matic chamber, at 21°C, under a light intensity of 40 µmol m-2 s-1 and a 12-hr photoperiod. After four weeks, the rooted microplantlets were transferred to ex vitro conditions. The plantlets were transferred to the greenhouse into pots with substrate containing both sterilised peat and sand in a ratio of 2:1. After five weeks, the plants were transferred to the field and their morphological stability was evaluated. Cryopreserved chicory lines The cryopreservation procedure, optimized for ‘Rosso di Chioggia’ shoot tips, was then tested on shoot tips from in vitro plants of three red chicory lines, ‘Rosso di Treviso precoce’ (TVP S5), ‘Rosso di Treviso tardivo’ (TVT) and ‘Variegato di Castelfran- co’ (C90 S6’). All three lines are included in a pro- gram to safeguard and conserve red chicory in the Veneto region. Encapsulation-vitrification procedure ‘Rosso di Chioggia’ shoot tips, cold-hardened at 4°C for two days, were encapsulated into 2% Na-algi- nate beads and treated with LS solution for 1 hr on a rotary shaker at 25°C. When this solution was removed, the beads were dehydrated by exposure to PVS2 for 2, 3 or 4 hr at 0°C, then placed in cryovials (five beads per cryovial) and plunged immediately into LN. Encapsulated shoot tips were thawed in a waterbath at 40°C for 3 min and placed on MS prolif- eration medium. Survival was assessed after three weeks. Data analyses A minimum of 30 samples were used for each treatment and each experiment was repeated twice. Statistical analysis of percentages was carried out by the χ2 test or non-parametric statistical test, the Post Hoc Multiple Comparison Test (Marascuilo and McSweeney, 1977), both at P ≤ 0.05. Genetic stability assessments of cryopreserved plantlets Random Amplified Polymorphic DNA (RAPD) analysis was applied on red chicory in vitro shoots to evaluate the genetic stability of cryopreserved and non-cryopreserved shoots (untreated control) collect- ed from the same stock cultures. Genomic DNA was extracted from approximately 100 mg of leaves with a ‘DNeasy Plant Mini Kit’ (Quiagen). DNA concen- tration was determined using a spectrophotometer at 260 nm, and an aliquot of DNA was diluted to a work- ing concentration of 20 ng µl-1. RAPD profiles were generated using 24 arbitrary 10-mers as primers, of which 10 primers were then selected for the reproducibility, the legibility and the 47 stability of the RAPD pattern: 1253, 1247, M2, M3, M10, M13, A1, A5, A9 and C7 (Table 1). The amplification of DNA was performed accord- ing to Vettvori et al. (1996). Fragments were separated on 2% agarose gels by electrophoresis and visualized by ethidium bromide staining under UV light. For each primer, amplification reactions were repeated at least twice and only those having reproducible band partners were used. Minor fragments, which tend to be unstable in staining intensity, and therefore not reliable, were not considered. 3. Results Influence of PVS2 times and preculture treatments Prolonged exposure at 0°C to the vitrification solu- tion appeared to be harmful for explants. Indeed, shoot- tip survival decreased after exposures of 90 min or more, while 30-min treatment was not enough to pro- tect the explants during ultra rapid freezing (Table 2). When the incubation time was limited to 60 min, more than 72% of cryopreserved (+ LN) shoot tips survived after plating onto the proliferation medium (Fig. 1c). Incubation for 90 and 120 min in the PVS2 solution showed even a slight decrease in percentage survival of shoot tips without freezing (- LN). This result high- lights the importance of incubation time with vitrifica- tion solution for shoot tip survival. To enhance the shoot tips’ osmotolerance to the vit- rification solution, preculture treatments with different sucrose concentrations for two days at 4°C was applied before the LS and PVS2 treatments. However, frozen explant survival exhibited a significant decline when the shoot tips were pre-cultured on media containing high sucrose concentrations (Table 3), indicating that sucrose treatments are not appropriate to improve red chicory shoot tip survival. All three red chicory lines can be successfully cry- opreserved by loading shoot tips in PVS2 for 60 or 90 min, prior to freezing in LN. Among the lines, maxi- mum explant survival ranged between 65% in ‘Rosso di Treviso precoce’ and 76% in ‘Rossso di Treviso tar- divo’ and ‘Variegato di Castelfranco’, respectively with 60 and 90 min of PVS2 treatment (Table 4). Recovery media The addition of BA to the recovery medium was found to be beneficial for post-thaw recovery of the shoot tips, even if with an increase of the concentration callus formation is stimulated. The highest post-thaw survival of ‘Rosso di Chioggia’ shoot tips was obtained with 0.5 µM BA (79%) (Table 5). When 1 µM TDZ was used, 33% of shoot tips survived, but after the first subculture (21 days), they stopped growing, after which no shoots developed. There was no survival of shoot tips on hormone-free medium supplemented with activated charcoal. Table 1 - Nucleotide sequences of DNA primers used for RAPD analysis Primer Primer sequence 1253 1247 M2 M3 M10 M13 A1 A5 A9 C7 GTT TCC GCC C AAG AGC CCG T ACA ACG CCT C GGG GGA TGA G TCT GGC GCA C GGT GGT CAA G CAG GCC CTT C AGG GGT CTT G GGG TAA CGC C GTC CCG ACG A Table 2 - Survival of cryopreserved shoot tips of ‘Rosso di Chioggia’ after exposure to PVS2 for different time periods. Data were recorded three weeks after thawing (LN, liquid nitrogen) PVS2 exposure time (min) Shoot tip survival (%)(z) 30 60 90 120 100 a 100 a 80 b 80 b - LN + LN 33.5 b 72.5 a 40.0 b 30.0 b (z) In each column, percentages followed by different letters are signi- ficantly different at P ≤ 0.05 by the post hoc Multiple Comparison test. Table 3 - Effect of sucrose concentration in preculture medium (two days at 4°C) on ‘Rosso di Chioggia’ shoot tips cryopreserved following 60 min PVS2 treatment Sucrose concentration (M) Shoot tip survival (%)(z) 0.09 0.3 0.7 68.0 a 47.5 a 17.5 b (z) Percentages followed by different letters are significantly different at P ≤ 0.05 by the post hoc Multiple Comparison test. Table 4 - Shoot tip survival percentage of three selected red chicory lines, following incubation and immersion in LN Red Chicory Line Loading time with PVS2 (min) (z) For each select line and each column, percentages followed by dif- ferent letters are significantly different at P ≤ 0.05 by the χ2 test. Shoot tip survival (%)(z) ‘Rosso di Treviso precoce’ ‘Rosso di Treviso tardivo’ ‘Variegato di Castelfranco’ - LN + LN 60 90 60 90 60 90 80.0 a 70.0 a 80.0 a 90.0 a 90.0 a 100 a 65.0 a 55.0 a 66.7 a 76.7 a 50.0 a 76.0 b Table 5 - Effect of recovery media on ‘Rosso di Chioggia’ shoot tip sur- vival (three weeks) and regrowth (six weeks) after thawing Recovery medium 0.5 µM BA 1.0 µM BA 5.0 µM BA 1.0 µM TDZ 0.5 g/l AC 79.1 a 47.6 b 47.8 b 33.3 c 0 Shoot tip survival (%) Shoot tip regrowth (%)(z) 100 a 95.0 a 95.4 a 0 -- (z) In each column, percentages followed by different letters are signi- ficantly different at P ≤ 0.05 by the post hoc Multiple Comparison test. 48 A further increase of regrowth was easily achieved by transferring the explants in tubes containing prolif- eration medium with BA. The best concentration for the development of ‘Rosso di Chioggia’ chicory shoots was again 0.5 µM. Indeed, the plantlets obtained from this cytokinin treatment were generally taller and pro- duced more shoot apices (Fig. 1d). After in vitro root- ing (Fig. 1e), the microplantlets were transplanted into pots and successfully acclimated in vivo (Fig. 1f). Field observations of the plantlets confirmed the morphological stability of acclimatized plants with the original mother plants (Fig. 2). Vitrification vs encapsulation-vitrification Cryopreservation by shoot-tip vitrification, using the PVS2 solution, was compared with the encapsula- tion-vitrification procedure in ‘Rosso di Chioggia’ line. The highest survival percentage of cryopreserved shoot tips was obtained by vitrification procedure (Table 6). Fig. 2 - Experimental field, the arrow indicates the plot with cryopre- served ‘Rosso di Chioggia’ plants. Cryogenic procedure Shoot tip survival (%)(z) Vitrification Encapsulation - vitrification 76.0 a 35.2 b (z) Percentages followed by different letters are significant at P ≤ 0.05 by the post hoc Multiple Comparison test. The encapsulation-vitrification procedure proved to be less effective for the cryopreservation of the selected red chicory line. However, only a maximum of 35% explant survival was achieved when the longest treatment of the beads with PVS2 was applied; this percentage is marked- ly lower than that reported in the literature using an encap- sulation-dehydration procedure in chicory (Vandenbuss- che et al., 1993). Assessment of molecular stability by RAPD To assess the genetic fidelity of plantlets regrown from cryopreserved shoot-tips, the RAPD patterns were com- pared with untreated samples of the same red chicory lines. Out of 24 primers screened, 10 selected primers pro- duced clear and reproducible bands. The number of bands for each primer varied from five to twelve. Each primer generated a set of amplification products of a size ranging between 350 bp and 3000 bp. Preliminary results proved that the genetic fidelity of the cryopreserved lines was maintained. For all 10 primers tested, RAPD fragment patterns of plantlets from cryopreserved shoot-tips did not show differences with respect to untreated shoots. Figure 3 represents amplified band patterns produced by two primers (1253 and 1247). Genetic fidelity was confirmed also by the other primers. 4. Discussion and Conclusions ‘Rosso di Chioggia’ shoot-tips cryopreserved using LS treatment for 30 min, PVS2 for 60 min at 0°C, and recov- ery on medium containing 0.5 µM BA resulted with a high survival rate (79%). This result is slightly inferior to that obtained in Belgian endive, cv. Flash (83%) where Demeulemeester et al. (1992) used a more complex pro- cedure, consisting of cooling the explants to -40°C at a rate of 0.5°C min-1 prior to immersion in LN. In the pre- sent study, the vitrification protocol applied to different red chicory lines showed high survival percentages, even if tolerance to the PVS2 varied in different lines. Differ- ences among lines can be considered to be genotype- dependent; these results are consistent with other experi- ences on cryopreservation in other plant species (De Bou- caud et al., 2002; Kim et al., 2006; Benelli et al., 2009). Table 6 - Shoot tip survival percentage of ‘Rosso di Chioggia’ shoot- tips after two different cryogenic procedures Fig. 3 - RAPD profiles generated with primers 1253 (left) and 1247 (right) from untreated and cryopreserved plantlets of ‘Rosso di Chioggia’ line. In both images, lanes M correspond to the 100-bp ladder; lanes 1-9 correspond to the amplification products from untreated sam- ples, lanes 10-17 correspond to the amplification products from cryopreserved plantlets. 49 In many cases, preculture with sucrose can be very efficient to improve cryopreservation (Matsumoto et al., 1998), but such treatments applied on apical buds of several species did not induce increases in recovery after vitrification (Sakai, 2000). Sugar treatments can influence the membrane and protein composition (Ramon et al., 2002; Carpentier et al., 2005), influenc- ing flexibility and permeability of the membrane. In red chicory shoot tips, preculture treatment on MS with 0.09 M sucrose for two days before PVS2 loading resulted in the best survival with respect to 0.3 and 0.7 M sucrose, but it did not improve the post-thaw sur- vival percentage in the cryogenic procedure. The type and concentration of growth regulators in the recovery medium is important for survival and regrowth of cryopreserved explants (Turner et al., 2001). Red chicory shoot tip survival (79%) was improved when the recovery medium contained 0.5 µM BA; callus formation was induced when the BA concentration was increased, in particular with 5 µM BA. Callus is not desired and represents a limitation in development of the cryopreserved explants. This phe- nomenon is observed in several species when a high concentration of BA was used (Wang et al., 2003). Demeulemeester et al. (1993) held the explants for one week on medium supplemented with plant growth reg- ulators and then transferred them to hormone-free medium to avoid a callus phase in three varieties of chicory (Flash, Rumba and Carolus) after cryopreser- vation. This protocol was effective for restricting callus formation, but led to a decrease in survival percentage. In the present study, with the ‘Rosso di Chioggia’ line, we obtained the highest survival rate without callus formation by applying the lowest BA concentration and the best regrowth when the single cryopreserved shoot was transferred in tube. Addition of activated charcoal in post-thaw recov- ery medium resulted deleterious for shoot tip survival and regrowth; whereas thidiazuron gave a low survival of explants after three weeks but no subsequent growth. Rooted microshoots of ‘Rosso di Chioggia’, obtained from cryopreserved shoot-tips, exhibited high survival and vigour in the greenhouse. Application of the encapsulation-vitrification proce- dure showed a limited survival of cryopreserved explants and further experimentation is needed to opti- mize the protocol for these red chicory lines. A few genetic studies using molecular markers have been carried out on red chicory, mainly for the genetic characterization of commercial varieties (Barcaccia et al., 2003), and particularly RAPD markers were used to construct the genetic map of C. intybus (De Simone et al., 1997) to identify and to evaluate the phylogenet- ic relationships among cultivars of chicory (Koch and Jung, 1997; van Stallen et al., 2000, 2001; Barcaccia et al., 2003). Assessment of the genetic stability of cryopreserved shoots can be performed with different techniques (Harding, 2004). In this study, RAPD markers were adopted because of their simplicity, rapidity and ability to screen a randomly large part of the genome. Genetic stability was reported after RAPD analysis of cryopreserved plantlets and the untreated shoots. Comparison of the DNA patterns of control and frozen material did not reveal any variations caused by the cryoprotectant treatments or cryostorage. 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