A validated slow-growth in vitro conservation protocol for globe artichoke germplasm: A cost-effective tool to preserve from wild to elite genotypes

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A validated slow-growth in vitro conservation protocol for globe artichoke germplasm: A cost-effective tool to preserve from wild to elite genotypes Raffaela Tavazza a,∗ , Nestor Alonso Rey b , Velia Papacchioli a , Mario Augusto Pagnotta c a

ENEA, CR Casaccia, Unit of Sustainable Development and Innovation of the Agro-Industrial System, Via Anguillarese 301, 00123 Rome, Italy Big Heart Seed Company, 1280 Main Street, Brawley, CA 92227, USA c Department of Science and Technologies for Agriculture, Forestry, Nature and Energy (DAFNE), University of Tuscia, Via S. C. de Lellis snc, 01100 Viterbo, Italy b

a r t i c l e

i n f o

Article history: Received 12 March 2015 Received in revised form 13 September 2015 Accepted 18 September 2015 Available online xxx Keywords: Cynara cardunculus var. scolymus L. Ex-vitro performance In vitro storage Molecular marker Morphological trait

a b s t r a c t A reliable and reproducible genotype-independent protocol for slow-growth storage of globe artichoke was established for the first time to meet two needs: genetic resources conservation and labour costs reduction in commercial laboratories. Growth reduction was achieved by supplementing osmotic agents to the media. Plant responses to in vitro storage, genetic stability and field performance were the parameters used to evaluate the germplasm conservation conditions. Forty-nine treatments were applied, as the result of seven genotypes with seven media. After 12 months of storage, culture survival across genotypes ranged from 65% to 85% and all the media tested supported 100% regrowth. All genotypes regained their full growth potential within two months. Genetic stability between mother plants grown in the field and in vitro conserved plantlets at 6 and 12 months of storage was assessed by molecular markers to identify the most suitable storage media. Protocol suitability was validated by a field test using an approved list of plant descriptors for globe artichoke. Morphological data highlighted that the slight genetic instability detected by molecular markers did not affect significantly plants morphology and their agronomic traits. The results indicate that the minimal growth medium of choice for globe artichoke conservation is the one where the seven genotypes displayed phenotypes similar to the control, coupled with the lowest percentage of changes (2.43%) at a molecular level. As far as we know there are no published reports on in vitro conservation protocolfor globe artichoke applied to different genotypes and validated by an appropriate field test. © 2015 Published by Elsevier B.V.

1. Introduction Native to the Mediterranean basin, the globe artichoke (Cynara cardunculus var. scolymus L.) is widely grown around the world. Italy and Spain are the world’s leading producers followed by France (FAO STAT 2012; http://faostat.fao.org). Italy harbours the richest primary cultivated artichoke gene pool, and houses the most abundant in situ diversity (Bianco, 1990).

Abbreviations: ANOVA, one-way analysis of variance; IBA, indole 3 butyric acid; ISSR, inter-simple sequence repeats; KIN, kinetin; NAA, 1-naphthaleneacetic acid; PIC, polymorphic information content; SSR, simple sequence repeats. ∗ Corresponding author. E-mail address: [email protected] (R. Tavazza).

Globe artichoke is the third largest vegetable crop cultivated in Italy, after tomato and potato. Interest in this species has recently increased along with its new possible uses. Globe artichoke is commercially important for its dietary and pharmaceutical value (Saènz Rodriguez et al., 2002; Coinu et al., 2007; Fantini et al., 2011). It is also exploited for oil production from seeds (Foti et al., 1999), inulin from roots (Raccuia and Melilli, 2010) as well as for energy from biomass (Ierna and Mauromicale, 2010; Ierna et al., 2012). It is thus crucial to gain access to a wide range of plant genetic resources that provide varied traits. As globe artichoke is a highly heterozygous species, it leads to a segregating progeny and considerable variation in seed types. As a result, its germplasm cannot be stored effectively by conventional means. To preserve the genetic integrity, selected genotypes are maintained vegetatively (Barbieri, 1967; Snyder, 1979) and

http://dx.doi.org/10.1016/j.scienta.2015.09.024 0304-4238/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Tavazza, R., et al., A validated slow-growth in vitro conservation protocol for globe artichoke germplasm: A cost-effective tool to preserve from wild to elite genotypes. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2015.09.024

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propagated as genotypes Traditionally, the field collection of globe artichoke is the most widely used storage method, and provides the possibility of continuously characterizing and evaluating the germplasm. Although field collection provides easy access to conserved material, its maintenance is time-consuming, it requires a lot of space and is labour-intensive. In addition, losses of germplasm maintained in the field collection can occur due to environmental and biological hazards (Babes et al., 2004; Bianco, 1990). Nowadays, only limited amounts of artichoke germplasm are conserved in field collections, which are mainly carried out by public institutions. Strict international rules have established the sanitary and qualitative requirements for safe germplasm exchange [Directives 93/61/CEE and 93/62/CEE (OJ L250, 07/10/1993 pp. 19–30)]. Therefore, the exchange of plant material is becoming increasingly difficult due to the great risk of transferring disease. Alongside field practices, propagation through in vitro meristem culture has proved to be a safe and more practical method for the collection, multiplication and conservation of valuable globe artichoke germplasm. This technique also leads to the production of large-scale, phenotypically homogeneous and disease-free plants, in particular for spring cultivars (De Leo and Greco, 1973; Ancora et al., 1981; Pecaut and Dumas de Vaulx, 1983; Rossi and De Pauli, 1992). In Italy, most commercial cultivars are the result of the in vitro isolation of selected individuals during propagation (Ancora, 1986; Ancora and Saccardo, 1987). The extensive use of this propagation method has led to the conservation of genetic resources, as well as the large-scale production and commercialization of superior genotypes by private tissue culture laboratories. More than four million micropropagated artichoke plants were produced in Italy in 2010, to provide propagation material to farmers (Lambardi and Previati, 2012). This has led, over time, to both the substitution of local genotypes for improved uniform varieties and changes in growing techniques. In turn, it causes the possible erosion of globe artichoke genetic resources and the loss of valuable material. Since 2007, artichoke landraces have been collected, characterized and preserved in order to conserve these gene pools both in field and in vitro collections (CYNARES EU project, Pagnotta, 2012). The high rates of multiplication achieved by micropropagation and the frequency of sub-cultures do not match well with the management of large in vitro collections as this would result in increased costs and the risk of contamination leading to losses of genetic material (Rey et al., 2013). Micropropagation has thus been considered only for the short-term storage of globe artichoke germplasm. Various in vitro conservation methods can be employed, depending on the use and storage duration required (Engelmann, 1997, 2011). To establish globe artichoke gene-bank facilities, the two most widespread strategies are slow-growth systems and cryopreservation. Cryopreservation was recently carried out for globe artichoke (Tavazza et al., 2013), and can be used for the long-term conservation of germplasm, but not for its distribution. The establishment of a medium-term conservation protocol would be useful for international germplasm distribution and, as a complementary option, for the safer conservation of the globe artichoke germplasm (Withers and Engelmann, 1998). This method of storing the germplasm could also be beneficial in assisting the micropropagation industry to minimise the costs of commercial under-production. In fact, in a commercial tissue culture laboratories there is no year-round demand for young plants but there are peaks in delivery and labour. Our result are particularly innovative since in terms of the current state of knowledge, only two papers have reported the slow-growth conservation of globe artichoke (Bekheet, 2007; Benelli et al., 2010), while to the best of our knowledge no data on the field performance of globe artichoke plants after retrieval from storage conditions are available.

The main purpose of ex situ gene-banks is to maintain the integrity and functionality of stored samples. The maintenance of the true-to-type nature of in vitro plants is an important requisite to uphold certain agronomic and horticultural traits when using elite genotype, not just for conservation, but also for commercial purposes. Genetic stability in tissue culture has long been a concern in the application of in vitro techniques for the conservation of crop germplasm. This is why there is great interest in techniques that can determine whether the material retrieved from in vitro conservation is genetically identical to the material accessed. Molecular markers are being increasingly used to monitor genetic variations and germplasm identification (Mondini et al., 2009). For conservation purposes they have been efficiently used to detect genetic stability in potato by AFLP (Aversano et al., 2011), in Hydrangea macrophylla by ISSR (Inter-Simple Sequence Repeats) (Liu et al., 2011), and in guava and globe artichoke by both SSR (Simple Sequence Repeats) and ISSR markers (Rai et al., 2012; Rey et al., 2013). The number of molecular markers available in globe artichoke has increased considerably and we successfully set a series of feasible markers to be used to characterize globe artichoke in order to accurately discriminate between genotypes (Crinò et al.,2008; Boury et al., 2011; Ciancolini et al., 2012; Rey et al., 2013). The aim of this study was to devise a simple and reliable method for the medium-term storage of globe artichoke cultures by slowing down growth by osmotic stress without altering the germplasm’s genetic stability. The best storage parameters for slow growing artichoke germplasm were defined in terms of their genetic and field performance once retrieved from storage. 2. Material and methods 2.1. Plant material Seven Italian spring Romanesco globe artichoke genotypes were considered: S2, S3, S8, S11, S17, S18, S23. These were selected from a set of 19 agronomically interesting genotypes isolated from traditionally cultivated landraces (Crinò et al., 2008) and subsequently characterized (Ciancolini et al., 2012). Shoot explants, 2–3 cm in length, were collected from 6-year-old in vitro plants from a germplasm collection kept through monthly subculture on a Gik basal medium (Tavazza et al., 2004), supplemented with 2 mg l−1 Kinetin (KIN), 0.1 mg l−1 indole-3-butyric acid (IBA), 3% sucrose and 7 g l−1 Plant agar, pH 5.8 and maintained at 18 ± 2 ◦ C under a 16-h light photoperiod provided by cool-white fluorescent lamps (37.5 ␮E m−2 s−1 ). The same environmental conditions were applied in slow-growth experiments. For each genotype, plantlets were further multiplied by axillary branching at 4–5 week intervals to establish stock cultures. 2.2. In vitro storage conditions To slow down the growth of in vitro plants, thus extending the time between subcultures, the carbohydrate level in the Gik medium was increased by replacing 3% sucrose with different combinations and concentrations of sucrose plus mannitol or sorbitol. Altogether two concentrations of sucrose (2 and 3% w/v) with either mannitol (4% w/v) or sorbitol (2 and 4% w/v) were tested. Forty-nine treatments were applied, as the resulted combination of seven genotypes with seven media. Explants (about 1 cm long) from micropropagated plants were: (a) maintained through monthly sub-culture on Gik medium as a control, hereafter referred to as medium T0; (b) stored on Gik medium (T1) or on modified Gik medium containing 30 g l−1 sucrose and 40 g l−1 mannitol (T2); 30 g l−1 sucrose and 40 g l−1 sorbitol (T3); 30 g l−1 sucrose and 20 g l−1 sorbitol (T4); 20 g l−1 sucrose and 40 g l−1 sorbitol (T5); or

Please cite this article in press as: Tavazza, R., et al., A validated slow-growth in vitro conservation protocol for globe artichoke germplasm: A cost-effective tool to preserve from wild to elite genotypes. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2015.09.024

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20 g l−1 sucrose and 20 g l−1 sorbitol (T6). Each genotype/medium treatment was tested in four replicates, each consisting of six explants. The culture was performed in 300 ml glass jars with 40 ml of medium and stored for 6 and 12 months under standard culture conditions as noted above.

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acquisition, and read using Genemapper v 3.7. All ISSR fragments in the size range 50–500 bp were assumed to represent a single dominant locus, while for SSR, fragments in the range 200–250 bp were considered and were registered as a single co-dominant locus. All the reactions were performed twice and only the reproducible peaks were considered.

2.3. Survival and regrowth of stored cultures The effect of increased carbohydrate levels on culture survival (%) was rated at the end of both storage periods, i.e. 6 and 12 months, and compared to those grown on the Gik medium with or without the subculture (T0 and T1, respectively). Shoot length was measured at the end of the longer storage period. After retrieval from storage (6 and 12 months), shoots, for each genotype and each medium, were cleared of the dead parts and transferred onto the Gik medium under standard culture conditions for recovery assessment. The regrowth capacity in terms of percentage of shoots that were able to resume normal growth was evaluated 10 days after subculturing. The multiplication rate (number of shoots/explant) was recorded at the end of the first multiplication cycle under standard conditions (four weeks). Following the first cycle of multiplication the plantlets recovered at the end of the second six months of storage were sent to an industrial micropropagation laboratory (Invitroplant Italia srl-Cesena-Italy) for massive multiplication. 2.4. Rooting and acclimatization After four multiplication cycles (four weeks each), proliferating shoots (3–4 cm in length) were transferred individually onto root induction medium, i.e. basal Gik medium, supplemented with 2 mg l−1 1-naphthaleneacetic-acid (NAA), 3% sucrose and 7 g l−1 plant agar, pH 5.8. Rooting response was evaluated after six weeks and expressed as a rooting percentage. The rooted plantlets, regenerated after 12 months storage, were rinsed with tap water to remove traces of agar, and transferred into hydrated jiffy peat pellets. They were then kept in closed plastic boxes under high humidity and moderate light, for an initial acclimatization; the humidity percentage was then gradually decreased. The plantlets were subsequently potted in soil and hardened in the greenhouse, before being transferred to the field. 2.5. DNA extraction and marker analysis For each genotype, several plant materials were analysed: (i) leaves of field-grown mother plant (M) as point zero; new leaf tissue per genotype and treatment 10 days after retrieval from storage at (ii) 6-months and (iii) 12-months. After each sampling step and genotype leaves, randomly collected, were stored at –80 ◦ C until used. Total genomic DNA was extracted from 100 mg of frozen tissue, using the Gene MATRIX Bio-Trace DNA Purification Kit (EURxhttp://www.eurx.com.pl/ow userfiles/plugins/wysiwygeditor/ images/2/basic-en260814.pdf). DNA quality was checked by electrophoresis of the samples on 1% agarose gel and staining with ethidium bromide. DNA concentration was determined through spectrophotometry (Gallagher and Desjardins, 2007). The molecular analysis was run using four ISSR and four SSR primers. ISSR primers were selected from the University of British Columbia (Canada) primers list, as reported in Table 1. SSR primers (Table 1) consisted in two microsatellite loci isolated from C. cardunculus L. var. scolymus: CLIB02 and CLIB12 and two found in database accessions: CDAT01 and CDAT03 (Acquadro et al., 2003). The PCR reactions were performed as described by Rey et al. (2013). The forward primers, of both marker typologies, were labelled either with FAM or with HEX dyes. PCR amplicons were resolved using an ABI 3130xl sequencer machine (Applied Biosystem), automatic

2.6. Morphological analysis Field experiments were conducted at the experimental field station of ARSIAL (Latium Regional Agency for the Development and the Innovation of Agriculture) in Cerveteri Rome-Italy (41◦ 59 N 12◦ 01 E). Only hardened plants, which had been stored for 12 months, were transplanted to the field at the end of August 2012 with 2 × 2 m plant-to-plant and row-to-row distances in a completely randomized block design, with three replications. Six plants per genotype/treatment were evaluated agro-morphologically during spring 2013. Each plant was phenotyped, using 47 UPOV (International Union for the Protection of New Varieties of Plant) descriptors for globe artichoke, as described by Pagnotta et al. (2013). We also used a well-defined group of complementary Romanesco type descriptors validated in a previous work (Crinò et al., 2008; Ciancolini et al., 2012). Both groups of descriptors are widely used as morphological markers to characterize globe artichoke landraces in the field. These included qualitative traits (such as head colour and shape, and spiny presence) and quantitative traits (such as plant high, head yield, and leaf length). The quantitative traits were measured biometrically rather than by score, as UPOV prescriptions, since these are easier to record and are less affected by personal interpretation.

2.7. Statistical analysis Data for survival of shoots recovered from slow-growth culture were analyzed by ANOVA and presented as mean ± standard error (S.E.). Statistical differences among means were performed by the Duncan test; significance was accepted at the P ≤ 0.05 level. Before statistical analyses run by SPSS software (version 14.0), the data measured in percentages were transformed into arcsine square roots and those regarding the multiplication rate, into square roots. Differences in marker profiles were recorded between mother plants (M) and genotypes stored for 6 and 12 months on the different media (T). The percentages of changes found with both markers (SSRs and ISSRs) were computed and reported separately for the first and second six months for each media over genotypes. The possible bands for each ISSR primer (between 50 and 500 bp) were recorded. Each significant peak was assumed to represent a single locus with two possible alleles: band presence recorded as 1, and band absence recorded as 0. The resulting 1/0 matrix was used in the statistical analysis. Genetic data were analyzed by Genetic Data Analysis (GDA) software (Lewis and Zaykin, 2001) to obtain Nei’s genetic distance (Nei, 1972), percentage of polymorphism, and expected heterozygosity and Polymorphism Information Content (PIC) for each marker. PICs were measured as suggested by Botstein et al. (1980). Comparisons between results on plants conserved after 6 months versus 12 months were tested by the t-test (SPSS software). The morphological traits were analyzed by ANOVA, using the Generalized Linear Model procedure (GLM)—SPSS software version 14.0. Mean separations were performed by the Duncan test. Significance was accepted at the P ≤ 0.05 level. In order to develop a genotype-independent protocol for globe artichoke germplasm conservation, deliberately only data averaged over genotypes were considered and presented hereafter.

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Table 1 Effect of storage medium and length of storage (6, 12 months) on survival, multiplication rate, rooting and shoot length. 6 Months

12 Months

Storage media

Survival (%)

Multiplication rate1

Rooting (%)

Survival (%)

Multiplication rate1

Average shoot length (cm)

T0 T1 T2 T3 T4 T5 T6

100.0d ± 0.00 85.1a ± 3.84 95.8bc ± 2.41 98.2cd ± 1.24 87.5a ± 1.57 96.4bc ± 1.68 94.0b ± 2.71

4.0c ± 0.37 2.2ab ± 0.18 2.0a ± 0.29 2.3ab ± 0.25 2.6b ± 0.18 2.3ab ± 0.31 1.8a ± 0.13

46.3c ± 6.95 23.8a ± 6.57 21.1a ± 4.27 44.2bc ± 8.16 40.1bc ± 10.07 33.3b ± 6.49 42.9bc ± 9.80

100.0c ± 0.00 64.9a ± 9.23 85.1b ± 4.32 75.2ab ± 5.03 76.5ab ± 4.70 70.5ab ± 11.10 72.3ab ± 7.47

3.99e ± 0.37 1.98a ± 0.21 2.11a ± 0.17 2.29ab ± 0.27 2.95cd ± 0.25 2.64bc ± 0.30 3.41de ± 0.19

4.0c ± 0.31 1.6a ± 0.32 1.3a ± 0.15 2.1b ± 0.26 2.3b ± 0.18 2.2b ± 0.39 2.6b ± 0.30

1 Multiplication rate: mean number of shoots per explant after four weeks. Each value represents a mean ± SE of four replications with six explants. Data within columns followed by different letters indicate significant differences at P ≤ 0.05 by Duncan’s Multiple Range test.

Table 2 ISSR and SSR primers used, their sequence, annealing temperatures (Ta ), and PIC value. Primer

Sequence 5 → 3

Ta (◦ C)

PIC

ISSR

810 834 841 857

(GA)8 T (AG)8 YT (GA)8 YC (AC)8 YG

43 45 45 54

0.11 0.06 0.14 0.15

SSR

CDAT-01 CDAT-03 CLIB-02 CLIB-12

(TGA)5 T.A(TGA)5 T. (GTG)5 (GTG)5 (CA)11 (CA)4 GA(CA)7 (TA)4

55 57 55 54

0.4 0.45 0.16 0.36

Y = pyrimidine.

3. Results 3.1. Globe artichoke genotype responses to in vitro storage To slow in vitro culture growth for an extended period of time, modifications of the medium routinely used for the short-term conservation of globe artichoke germplasm collection were investigated. Culture survival at six months of storage ranged from 85% to 98%, decreasing to between 65% to 85% after 12 months (Table 1). For both storage periods, i.e. 6 and 12 months, the lowest survival was recorded when cultures were maintained in Gik medium without the subculture (T1). Conversely, the addition of osmotic agents, mannitol and/or sorbitol to Gik medium always resulted in an increased survival percentage for both storage periods, though with different percentages in the different media. The highest value (100%) was reached in the control cultures, maintained under standard grown conditions (T0). Note that the differences observed between the survivals of conserved-derived plantlets were limited. Measuring explant lengths at the end of 12 months highlighted the effectiveness of slow-growth treatments. All the storage conditions tested reduced, but did not completely halt, plant growth compared to controls, ranging in size from 1.3 cm (T2) to 2.6 cm (T9) vs. 4.0 cm (T0). Storing explants in Gik medium without sub-cultures (T1) or in Gik supplemented with mannitol (T2) resulted in a decrease in shoot length and vigor, whereas no significant differences in shoot lengths were found in response to the sucrose/sorbitol concentration used. As expected, in the absence of subculture, browning of outer leaves was observed in all genotype/media combinations during the storage periods. This was more pronounced in some media (T1, T2) than others (T5, T6). In any event, the inner parts of the plants remained green and viable (Fig. 1A and B). After retrieval from both storage periods (6 and 12 months), shoots, which were visually scored as viable, started to elongate when returned to standard culture conditions, resuming normal growth in 10 days (Fig. 2A). All the media supported regrowth of 100% up to 12 months. The effect of osmotic agents on shoot proliferation was assessed by calculating the multiplication rate (number of shoots/explant) after the first multiplication cycle

on Gik medium following regrowth. Multiplication takes place through the proliferation of axiliary buds (Fig. 2B). The mean number of shoots formed per explant depended mainly on the storage media used, while it was not significantly affected by genotype (data not shown). In addition, surprisingly T1 was the treatment with the lowest multiplication rate at 12 months. On the other hand, at 12 months, multiplication rates in T6 medium were similar to T0, while T5 and T4 performed better than T1. Root formation occurred directly from the base of shoots within five weeks after transfer to root induction medium in all genotypes, except for genotype S2 which required a longer time to root. Spontaneous rooting was observed in some genotypes/media combinations both during storage (6–12 months) and multiplication phases (data not shown). The rooting response (Table 1) of shoots originating from cultures stored for six months in T3, T4, T6 media was not statistically different from the control (T0), while storing in T1, T2 and T5 media significantly reduced rooting percentages. Rooting percentage was not measured at longer storage periods (12 months), because after the first cycle of multiplication recovered plantlets were sent to Invitroplant Italia srl (Cesena-Italy), an industrial micropropagation laboratory, for mass multiplication. Forty healthy rooted plants per genotype and per storage medium were successfully acclimatized to greenhouse conditions (85% survival). All plants retrieved from in vitro conservation exhibited a phenotype similar to those maintained under standard growth conditions and appeared to undergo normal development. 3.2. DNA marker characterization At 6 and 12 months, ISSR and SSR profiles from genotypes stored in different media (T) were compared with those obtained from their respective field growing mother plants (M). The primers that we used (four ISSRs and four SSRs) had different abilitites in detecting genetic variation. Some of these differences are due to the different marker typologies; as expected PIC values were different between types of primers and also within the same type. Among ISSRs, primer 841 amplified the maximum number of bands and showed, with primer 857, the highest PIC value (Table 2), whereas, primer 834, in a multi-band pattern, amplified the least. In the SSR

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Fig. 1. Slow-growth conservation of globe artichoke genotypes 12 months-old preserved explants grown in presence of (A) sorbitol or (B) mannitol.

Fig. 2. In vitro development of retrieved globe artichoke plantlets after 12 months of conservation. (A) Plant recovery on Gik medium after 10days (B) Proliferation of auxilary budson Gik medium after 4weeks.

Fig. 3. Cumulative percentage of changes (y-axis) in genotypes stored on different media (T0 to T6) (x-axis) for 6 and 12 months with respect to field grown another plants. Different letters indicate significant differences at P ≤ 0.05 by Duncan’s Multiple Range Test.

primers group, CLIB-02 had the lowest PIC value, while CDAT-03 had the highest. The mean percentages of changes occurring in each overall genotype storage media are reported in Fig. 3. Since changes were measured in relation to the mother-plants (M), the control (T0) also

showed variations at 6 and 12 months, although during this time the control was sub-cultured monthly. The results clearly indicated that at six months storage, the percentages of changes were higher than those occurring at the end of the second six months, regardless of the media used. In addition, the average over the genotypes highlighted that some media were better than others in terms of changes recorded. Comparing the seven storage media at 6 and 12 months highlighted that the average number of changes in T6 and T5 media were always statistically lower. After 12 months of storage, variations in the pattern of changes detected among media were as follows: T6 and T5 had the lowest change rates (2.43% from the mother plant), T1 and T2 showed no significant differences compared to the control (T0) with an average of 2.87% of changes, and T4 and T3 had the highest average number of changes reaching 3.46%. The media ranking regarding the percentage of changes were almost consistent after 6 and 12 months. The ranking at six months was T6, T5, T2, T0, T1, T3, and T4, while at 12 months it was T6, T5, T0, T1, T2, T3, and T4. Regarding the average Nei genetic distance (Nei,1972) between plant materials grown on different media, the distance between media was very narrow ranging from 0.0022 to 0.0103. Bearing in mind this narrow distance, T6 and T5 were closer to the control (T0). Regarding the distance between mother plants and plant materials conserved under different treatments, all were narrow. However,

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Table 3 Nei (1978) genetic distance matrix between genotypes conserved on different media (T) and mother plants (M).

T1 T2 T3 T4 T5 T6 M

T0

T1

T2

T3

T4

T5

T6

0.0089 0.0085 0.0103 0.0078 0.0071 0.0068 0.0089

0.0030 0.0031 0.0059 0.0052 0.0076 0.0094

0.0047 0.0058 0.0056 0.0076 0.0100

0.0031 0.0022 0.0078 0.0100

0.0023 0.0079 0.0092

0.0052 0.0092

0.0085

Table 4 Statistically significant differences in morphological traits observed between genotypes, retrieved from different storage media or conventionally micropropagated, after one year of culture in the field. P level

.001

.029

.009

.000

.002

.000

.001

Media

Plant height (cm)

Attitude(1–9)

Leaf length (cm)

Length of lateral head (cm)

Diameter of lateral head (cm)

Main head maturity (n. days from 1st Jan)

Lateral head maturity (n. days from 1st Jan)

T0 T1 T2 T3 T4 T5 T6

57.60a ± 1.46 65.95b ± 1.64 64.09b ± 1.51 63.10b ± 1.49 66.33b ± 1.49 63.05b ± 1.71 63.99b ± 1.53

3.07b ± 0.17 2.53ab ± 0.20 2.48a ± 0.19 2.76ab ± 0.18 2.43a ± 0.18 3.13b ± 0.20 2.58a ± 0.18

90.79a ± 1.54 92.19bc ± 1.70 95.49c ± 1.59 93.12bc ± 1.57 91.71a ± 1.55 87.89a ± 1.78 95.76c ± 1.63

7.22a ± 0.10 7.58bc ± 0.12 7.87c ± 0.10 7.45ab ± 0.10 7.53b ± 0.11 7.74bc ± 0.12 7.49ab ± 0.10

7.40a ± 0.11 7.85bc ± 0.14 8.14c ± 0.11 7.71ab ± 0.12 7.74b ± 0.12 7.78bc ± 0.13 7.82bc ± 0.12

96.53bc ± 0.95 98.68c ± 1.05 93.71a ± 0.97 96.60bc ± 0.96 94.93a ± 0.95 97.43bc ± 1.09 95.09abc ± 0.98

104.47a ± 0.89 107.55b ± 1.01 104.33a ± 0.91 105.86ab ± 0.89 103.67a ± 0.95 106.09ab ± 1.03 103.27a ± 0.92

Each value represents a mean ± SE of three replications with three plants. Different letters, in each column, indicate significant differences at P ≤ 0.05 by Duncan’s Multiple Range test.

plants grown in T6 were closer to the mother plants, while plants grown in T3 were the further from the mother plants (Table 3). 3.3. Morphological characterization Ninety percent of the plants retrieved from the in vitro slow-growth conservation survived after field establishment thus enabling a field test for morphological characterization. Morphological data were recorded from six plants per genotype/medium, using the 47 selected globe artichoke descriptors; out of these, only seven traits showed statistically significant differences among the media overall genotypes; i.e. plant height, leaf attitude, leaf length, size of lateral heads (length and diameter), and time of maturity (main and lateral heads) (Table 4). The plant height and diameter of lateral heads were statistically different from the control (T0) for all media tested. All stored plants showed a greater vigour. The remaining five traits showed differences depending on the storage media used. For instance, leaf length of stored-derived plants was generally longer compared to the control, except for plants grown in T4 and T5 media which did not differ from the control (T0). The sizes of lateral heads were generally bigger, but not in genotypes from T3 and partially (only for the length of the lateral head) from T6 media. Regarding main head maturity, only genotypes from T1, T3, T5 and T6 media were not statistically different from the control (T0). In any event, although the seven traits were statistically different, the levels of differences recorded were very narrow: about 8 cm for plant height, 4 cm for leaf length, less than half cm for head dimension, and 4 days for maturity time. 4. Discussion Genetic resources of globe artichoke in Italy are currently managed by field and/or in vitro collections. Both strategies have drawbacks and technical constraints that limit their efficiency and threaten their security (Maxted et al., 1997; Dullo et al., 2001; Engelmann and Engels, 2002). Hence, it has become essential to optimize storage systems in order to limit the factors that can lead to somaclonal variation in germplasm maintenance over the long

term. This can mainly be achieved by slow-growth systems and/or cryopreservation-based approaches. Our objective was to investigate the feasibility of establishing and running an active in vitro artichoke collection, under minimal growth storage using osmotic stress which ensures the integrity and functionality of the stored plants. Unlike previous protocols that are designed only for single genotypes, we wanted to discover one single protocol that could be used for preserving genetically diverse materials. We followed a multidisciplinary approach that enabled us to work backwards from the data of the morphological and molecular characterization of the retrieved plants, in order to identify the most suitable conservation medium. The results demonstrated that it is possible to store globe artichoke germplasm under slow-growth conditions. The method was effective in terms of the 12-months storage which requires no human effort and has no negative effects on culture survival. Optimum results were achieved with the modified Gik medium supplemented with 20 g l−1 sucrose and 20 g l−1 sorbitol (T6 medium). In fact, since the seven genotypes raised from this medium displayed phenotypes that were similar to the control, coupled with the lowest percentage of changes at a molecular level, this was considered as the minimal growth medium of choice for storaging globe artichoke germplasm. As far as we know there are no published reports on a medium-term storage protocol for globe artichoke, validated by an appropriate field test using an approved list of plant descriptors. In addition, this is the first time that this technique has been applied to different genotypes. In vitro slow-growth can be achieved by low temperature, osmotic stress (Grout, 1991; Withers, 1991) or a low concentration of nutrients (Engelmann, 1991a,b). In globe artichoke, this was previously achieved by combining a temperature reduction with the absence of light. The result of the above studies demonstrated that shoots could be stored for up to three months at 6 ◦ C (Bekheet and Usama, 2007) or four months at 4 ◦ C (Benelli et al., 2010) without any detrimental effect on the viability and regrowth of the shoots. However, a decline in both parameters was recorded thereafter when the duration of storage reached 6, 9 and 12 months

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(Bekheet and Usama, 2007). Moreover, as already highlighted in other species (Gopal et al., 2002), the decrease in temperature led to undesirable effects such as vitrification on plants under in vitro conservation. Despite this, the remaining plants were suitable for recovery and proliferation (Bekheet and Usama, 2007). By comparing the two slow-growth conservation strategies, i.e. slow storage temperature and osmotic stress, within the same study, Bekheet and Usama (2007) highlighted that globe artichoke shoot culture could be better stored in cold and dark conditions rather than under osmotic stress. However, their results were based on testing only a single concentration of sucrose (40 g l−1 ) in combination with a single concentration of mannitol (40 g l−1 ) or sorbitol (40 g l−1 ). In contrast, we tested different combinations/concentrations of sucrose, mannitol and sorbitol. After 12 months storage, all the media assayed promoted an acceptable restriction of plant growth, compared to the control (T0). This restriction reached values ranging from 65% to 85% depending on the storage treatment used. As expected, the worst overall growth features were recorded after, 12 months of storage, with shoots maintained in standard multiplication medium (Gik) with 3% sucrose (T1) without a subculture, whereas, over time all selected items, compared to the control, were improved by osmotic stress. Mannitol is well known to inhibit the culture growth of various species (Negash et al., 2001) improving survival in stored potato (Sarkar and Naik, 1998) and enset cultures (Negash et al., 2001). Likewise, in globe artichoke, we detected a similar trend of both parameters, growth and survival, by adding 40 g l−1 mannitol to Gik medium (T2), which subsequently led to a slightly lower rate of multiplication and a significantly lower rooting response. Conversely, plants grown with sorbitol tend to exhibit a significant incremental growth rate, regardless of the combinations/concentrations tested. This probably is due to the fact that sorbitol requires more time to be metabolized by the plants. No significant differences in plant survival were highlighted either among sorbitol or the other treatments. Transfer of the green inner part of the in vitro-stored shoots to the standard multiplication medium led to 100% re-growth irrespective of the cultivar and the treatment used. Their full potential of growth was regained within two months. Interestingly, although the preserved genotypes appeared differently in different storage media, they behaved similarly and developed vigorous plantlets once transferred to fresh medium. After 12 months’ storage, culture responses were observed in all treatments. However, plants stored in media containing sorbitol (T3, T4, T5, T6) showed better responses in terms of multiplication rate (12 months) and rooting percentage (6 months) compared to those stored in media containing mannitol. Notably, of the former, shoots stored in T6 medium showed a similar multiplication and root induction trend as the control cultures, when maintained under standard growth conditions (T0). No culture abnormalities were observed throughout and after minimal growth maintenance. Our results differ from those for globe artichoke (Bekheet, 2007), potato (Lopez-Delgado et al., 1998) and P. peltatum (Lata et al., 2010), where the use of osmotic agents led to a low percentage of morphological abnormalities. As small differences in storage temperature may lead to substantially different results among genotypes within a species (Capuana and Di Lonardo, 2013) likewise, the differences reported here in globe artichoke responses to osmotic stress storage, compared to the published findings (Bekheet and Usama, 2007), could be explained by the fact that a colder storage temperature such as 18 ◦ C would be more effective than 25 ◦ C (Bekheet and Usama, 2007) for the storage of globe artichoke cultures. The fact that the germplasm can be stored effectively for 12 months without subcultures at 19 ◦ C,

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means that our protocol has no cooling costs and does not require refrigerated infrastructures. Conservation at normal propagation temperatures provides better genetic stability for the germplasm (Harding, 1999), thus eliminating one of the inevitable physiological stresses imposed by the in vitro storage method affecting genetic stability (Westcott, 1981). Several strategies have been used to assess the genetic fidelity of in vitro raised plants. The reliability and efficiency of different molecular markers in identifying genome changes have been frequently discussed (e.g. Wang and Szmidt, 2001; de Vicente and Fulton, 2003; Pagnotta et al., 2009; Kirk and Freeland, 2011 de Vicente and Fulton, 2003; Wang and Szmidt, 2001; Pagnotta et al., 2009; Kirk and Freeland, 2011 Kirk and Freeland, 2011), these differences are summarized by markers PIC value. Molecular marker tools for characterizing and fingerprinting genotypes are widely used in several species including globe artichoke (Pagnotta and Noorani, 2014), as well as for assessing the genetic fidelity of globe artichoke during in vitro micropropagation (Rey et al., 2013). In our study, variations were observed when profiles of genotypes stored for 6 and 12 months were compared to those of field-derived controls. We found that the greatest percentage of changes occurred, in all media, during the first six months of storage rather than in the following months. This is probably due to the initial stress induced by the in vitro storage conditions (Rey et al., 2013); subsequently as the cultures ‘adapt’, the change rate declines,which favours the use of longer-term conservation. Taking into account the average over genotype, T0 was always (6 and 12 months) in a central position in terms of the percentage ranking of changes, thus demonstrating that the standard methods could be improved since some media more than others resulted in a reduction of the number of changes, notably media T5 and T6. Since no approach used to assess genetic fidelity can lead per se to a reliable data interpretation, it was important to verify whether the changes observed by molecular analysis translated into a phenotypic change in the field. To what extent was the correlation between molecular analyses and morpho/physiological traits straightforward was an important question raised in a previous work (Rey et al., 2013). To answer this question, globe artichoke morphological descriptors in field tests were used to assess the presumed variation of the plant. In addition, information on field performance was also necessary to assess the yield potential of globe artichoke genotypes retrieved from in vitro conservation. UPOV descriptors, which have been recently revised, set the traits compared with some varieties used as controls (for example, plant height could be scored as tall, medium or short). In assessing phenotypic variations in single genotypes, it is easier and more consistent to use metric measurements (i.e. plant high in cm). In fact, they are more accurate as they do not depend on subjective human interpretation and are easier to record. Metric measurements can also be easily used in statistical analyses (Pagnotta et al., 2013). Although the analysis of morphological data of field grown plants revealed slight morphological differences, these narrow ranges of values were statistically different due to the absence of variations within genotypes between reps. Consequently, these slight variations did not lead to phenotype variations in agricultural value traits. The absence of differences between replications with consequent higher differences between treatments is due to the low variations in the experimental field. The discrepancy between molecular and morphological data, could be explained by the fact that the molecular markers investigated in the genome region were not necessarily related to those coding for the morphological traits recorded. The level of genetic instability that was detected could be attributed to somaclonal variations due to stresses induced by in vitro practices.

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Ultimately, morphological characterization provided no evidence of phenotypic changes in the regenerated plants after 12 months of in vitro storage. This indicates that the slow-growth conservation exerted no adverse effects on morphology or yield of in vitro conservation derived globe artichoke plants and did not hamper the onset of production compared to conventionally propagated plants. In conclusion, this protocol can aid in vitro management of a germplasm collection and private tissue culture laboratories where less subculturing decreases the occurrence of somaclonal variations and reduces the costs for personnel, energy and production. The results presented highlight the importance of a multidisciplinary approach (Rani and Raina, 2000) to validate and propose a protocol prior to release; together with the importance of appropriately characterizing the accessions examined, not only for conservation purposes but also for commercialization. Acknowledgments The authors acknowledge the CYNARES (EC) No 870/2004, CAR-VARVI projects, which received financial support from the AGRI GEN RES Community Programme (European Commission, Directorate-General for Agriculture and Rural Development), Italian Ministry of Agricultural, Food and Forestry Policies and European founds, respectively. The authors are grateful to ARSIAL (Cerveteri, Rome-Italy) for providing field facilities. References Acquadro, A., Portis, E., Lanteri, S., 2003. Isolation of microsatellite loci in artichoke (Cynara cardunculus L. var. scolymus). Mol. Ecol. Notes 3, 37–39. Ancora, G., 1986. Globe artichoke (Cynara scolymus L.). In: Bajai, Y.P.S. (Ed.), Biotechnology in Agriculture & Forestry, 2. Crops Springer Verlag, Berlin, pp. 471–484. Ancora, G., Belli, M.L., Cuozzo, L., 1981. Globe artichoke plants from shoot apices through rapid in vitro micropropagation. Sci. Hortic. 14, 207–213. Ancora, G., Saccardo, F., 1987. Carciofo: nuove tecniche di propagazione. L’informatore Agrario 4, 53–55. Aversano, R., Di Dato, F., Di Matteo, A., Frusciante, L., Carputo, D., 2011. AFLP analysis to assess genomic stability in Solanum regenerants derived from wild and cultivated species. Plant Biotechnol. Rep. 5, 265–271. Babes, G., Lumia, V., Pasquini, G., Di Lernia, G., Barba, M., 2004. Production of virus free artichoke germplasm. Acta Hort. 660, 467–472. Barbieri, R., 1967. Aspetti agronomici della coltura del carciofo Proceedings of the 2nd International. In: Congress studies on Carciofo, Laterza, Torino. Bekheet, S., Usama, I.A., 2007. In vitro conservation of globe artichoke (Cynara scolymus L.) Germplasm. Int. J. Agric. Biol. 9 (3), 404–407. Bekheet, S.A., 2007. In vitro preservation of globe artichoke germplasm. Plant Tissue Cult. Biotech. 17 (1), 1–9. Benelli, C., De Carlo, A., Previati, A., Roncasaglia, R., 2010. Recenti acquisizioni sulla conservazione in vitro in crescita rallentata. Proceedings of ‘IX Giornate Scientifiche SOI’ Italus Hortus 17, 91. Bianco, V.V., 1990. Carciofo (Cynara scolymus L.). In: Bianco, V.V., Pimpini, F. (Eds.), Orticoltura, Patron Editore. Bologna, pp. 209–251. Botstein, D., White, R.L., Skolnick, M., Davis, R.W., 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32, 314–331. Boury, S., Jacob, A.-M., Egea-Gilabert, C., Fernández, J.A., Sonnante, G., Pignone, D., Rey, N.A., Pagnotta, M.A., 2011. Assessment of genetic variation in an artichoke european collection by means of molecular markers. Acta Hortic. 942, 81–88. Capuana, M., Di Lonardo, S., 2013. In vitro conservation of chestnut (Castanea sativa) by slow growth. In Vitro Cell Dev. Biol. Plant 49, 605–610. Ciancolini, A., Rey, N.A., Pagnotta, M.A., Crinò, P., 2012. Characterization of Italian spring globe artichoke germplasm: morphological and molecular profiles. Euphytica 186, 433–443. Coinu, R., Cart, S., Urgeghe, P., Mulinacci, N., Franconi, F., Romani, A., 2007. Dose-effect study on the antioxidant properties of leaves and outer bracts of extracts obtained from Violetto di Toscana artichoke. Food Chem. 101, 524–531. Crinò, P., Tavazza, R., Rey, N.A., Trionfetti Nisini, P., Ancora, G., Pagnotta, M.A., 2008. Recovery and characterization of Italian artichoke traditional landraces of Romanesco type. Genet. Resour. Crop. Evol. 55, 823–833. De Leo, P., Greco, B., 1973. Nuova tecnica di propagazione del carciofo: Coltura in vitro di meristemi apicali. In: Proceedings of the 3rd Congress International Studies on Carciofo, Laterza, Bari, pp. 657–665. de Vicente, M.C., Fulton, T., 2003. Using molecular marker technology in studies on plant genetic diversity. In: Learning Module. Illus. Nelly Giraldo, IPGRI, Rome,

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