Comparative study on in vitro micropropagation response of seven globe artichoke [Cynara cardunculus var. scolymus (L.) Fiori] cultivars: open-pollinated cultivars vs F1 hybrids

Globe artichoke [Cynara cardunculus var. scolymus (L.) Fiori] growing has gained commercial importance in recent years due to its consumption as food. It has also started to attract attention in pharmaceutics. Due to globe artichoke’s stated importance, growers need large amount of pathogen-free, healthy starting materials for production. Stated material will maximize the yield while minimizing the costs. Hybrid cultivars have uniform in height and maturity and could be harvested concurrently; on the other hand, an open-pollinated cultivar would have useful potential that could be smoothly produced locally at a lower cost. In vitro micropropagation enabling these goals as it serves large scale, fast, reliable and realistic alternative method to classic propagation via offshoots. The aim of the present study was to comparatively evaluate the micropropagation efficiency of two important local open-pollinated (OP) cultivars (‘Bayrampaşa’, ‘Sakız’) and five F1 hybrid cultivars (‘Olympus’, ‘Madrigal’, ‘Sambo’, ‘Green Globe’, ‘Imparator’), on the basis of total subcultures they were subjected to. Various plant growth regulators at various combinations were assessed for in vitro micropropagation and subsequent in vitro rooting. 3/4 basic MS medium supplemented with 0.05 mg L BA + 0.005 mg L IBA was determined as the best media combination for in vitro micropropagation, while 10.0 mg L IAA + 1.0 g L activated charcoal adding to 1/2 basic MS medium had positive effects on in vitro rooting. According to results, the micropropagation efficiency varied based on cultivar differences and number of subcultures regardless of being OP or F1 hybrid. The present study demonstrated that in vitro propagation of globe artichoke could be a valuable process for assessing mass propagation regardless of using F1 or OP cultivars. Considering the OP cultivars are cheap in terms of price in a comparison to F1 hybrid cultivars, OP cultivars could be also recommended to be used for in vitro mass propagation.

for its immature flowers (heads) has recently begun to be used in many fields, especially in pharmaceuticals, due to the high bioactive components of its body and leaves which are still being considered as waste material.
It can be produced both sexual and asexual. Sexual production is provided by seeds, asexual production refers to vegetative production. It is a cross-pollinated plant species and when propagated by seed, plants display high heterogeneity. Due to that it is generally not preferred for commercial production. Traditionally, vegetative propagation has become prominent (Bekheet et al., 2014). Offshoot or ovoli is used for vegetative propagation. However, vegetative propagation has some difficulties such as transferring some diseases and pests through the main plants used in the reproduction, the low reproduction rate during the growing period hinders genetic and agronomic development, physiological differences between shoots taken from the same parent plant and being less suitable for the mechanization (Ancora, 1986;Ordás et al., 1990;Morzadec and Hourmant, 1997;Rey et al., 2013;Saccardo et al., 2013;Bekheet et al., 2014;Campanelli et al., 2014;Dawa et al., 2018;Mazzeo et al., 2020).
In vitro methods help to overcome such problems and are seen as fast, reliable and realistic alternative methods that can be used for large scale production Campanelli et al., 2014). Among these in vitro methods, the micropropagation technique was first developed in globe artichokes by De Leo and Greco (1973), and is a powerful alternative to traditional propagation. Homogenous, free from diseases and pests, healthy plants can be obtained by in vitro micropropagation method, while this method also provides a high reproduction rate. In previous micropropagation studies shoot tip, meristem and seeds were used as explants (Ancora et al., 1981;Pécaut et al., 1983;Rossi and De Paoli, 1992;El Boullani et al., 2012;Bekheet et al., 2014;Dawa et al., 2018). Due to the infection problem encountered in shoot tip culture, researchers decided to use meristem culture in order to obtain healthy, virus and other pathogens free plantlets. Different cultivars need different kind of incubation practices as well as different media combination and plant growth regulators. If all stated parameters are optimized, healthy plants can be obtained and the micropropagation rate can be increased. Plants obtained by in vitro micropropagation method perform well in terms of both qualitative and quantitative properties in cultivation and therefore compensate high in vitro production costs (Saccardo et al., 2007). In addition to all of these positive properties, it is important to provide an opportunity for the selection of plants with the high medical value among plants that are rapidly propagated in vitro.
Growers believe that the key to success requires combination of various approaches. Due to F1 hybrids supplying a high degree of uniformity, disease resistance, and increased yield and "hybrid vigor", F1 hybrids are preferred by the majority of growers (Bonasia et al., 2010;Saccardo et al., 2013). On the other hand, it is also known that open-pollinated (OP) globe artichoke cultivars are prominent in some countries in the world as they are ideally adapted to certain environmental conditions. Due to the fact that OP's higher degree of genetic heterogeneity, they have high genetic diversity (Noorani et al., 2013;Mauromicale et al., 2018). Diminishing the possibility of disease or other genetic problems may help to protect from the environmental stresses. Additionally, it can be identified as a new trend that promoting some OP cultivars use among growers is encouraged by several governments in developing countries.
As stated above several in vitro studies were conducted on in vitro micropropagation of globe artichoke. To our knowledge, there is no study comparing the in vitro micropropagation responses of OP cultivars and F1 hybrids in globe artichoke. The present study was therefore conducted to reveal the in vitro micropropagation responses of 2 important local OP globe artichoke cultivars and 5 commercial F1 hybrid globe artichoke cultivars.

Culture preparation stage
For the initiation of micropropagation, young shoots, about 10-15 cm size, were used as explant. The shoots collected from the experimental field were first washed under running tap water and soak in antibacterial soap for 10 minutes (900 mL of purified water + 100 mL of antibacterial soap). Subsequently, these plant materials were subjected to 2-stage surface sterilization in a laminar air flow workbench. In the initial surface sterilization process, the plant materials were treated with 40% (4.5% sodium hypochlorite, v/v) commercial bleach solution for 15 minutes and then rinsed 3 times with sterile distilled water. After the first stage of surface sterilization, the plants' surface areas were reduced by cutting. Second surface sterilization stage was applied to these plant materials with 20% of commercial bleach solution (4.5% sodium hypochlorite, v/v) for 5 min and rinsed 3 times with sterile pure water. After successful 2-stage sterilization procedures, the meristems were separated from the main plant material by sterile forceps and scalpel and cultured in the medium prepared for micropropagation ( Figure 1). Figure 1. Separation of meristems from mother plant explants and culturing in micropropagation medium (bar = 1.0 cm) Murashige and Skoog (MS) (1962) was used as the basic medium for micropropagation. Two different media combinations were used in the study: ¾MS medium was supplemented with 0.05 mg L -1 BA + 0.005 mg L -1 IBA (No.1) and 0.25 mg L -1 BA + 0.025 mg L -1 IBA (No.2). These two media combinations were combined with sucrose (3.0%) and agar (6.0 g L -1 ) and pH was adjusted to 5.8 before autoclaving.

Meristem culture induction and axillary shoot proliferation
The meristems were initially cultured in test tubes containing 10 mL of medium with one explant per tube ( Figure 2). After 4 weeks, the shoots of the meristems were cut off to 1-3 cm length and transferred to 500 mL glass jars containing 100 mL medium. During subcultures, 5 explants were placed in each jar. Sub-culturing was repeated with 4 weeks intervals and continued until the 10 th subculture. Cultured plants were kept under 25±1 °C and 16/8 photoperiod conditions.
While the micropropagation was in progress, the acclimatization of in vitro rooted plantlets to the in vivo conditions was also carried out. For this purpose, in vitro rooted plantlets ( Figure 3) were transplanted into plastic trays containing peat: perlite (1:1) mixture. Plastic trays were covered with polyethylene cover during 10 days. The plantlets were watered twice a day. During 30-40 days of acclimatization process observations were made on plantlets and at the end acclimatization period surviving plants were transferred to the field.

Evaluated parameters
In vitro micropropagation rates, the average number of axillary shoots per explant, the average number of leaves per axillary shoot, in vitro root formation and respond to acclimatization were compared on the basis of being OP and F1 hybrid globe artichoke cultivars. Since each subculture was carried out with 4 weeks intervals, the number of new shoots was counted to calculate multiplication rate (MR) at the end of 4 weeks.

Statistical analysis
The experiment was performed in three replications for comparing 2 OP globe artichoke cultivars and 5 F1 hybrid globe artichoke cultivars to analyze their in vitro micropropagation response. Three jars were used in each replicate and 5 explants were used in each jar. The data obtained were subjected to variance analysis in the JMP package program and the differences between the averages were determined by LSD test.

Results and Discussion
Disinfection protocol Globe artichoke can be propagated via in vitro methods or traditional methods by using offshoots. In a comparison between in vitro propagation and traditional propagation methods, in vitro propagation has several advantages such as high multiplication rate and obtaining disease free starting material. But in vitro multiplication methods have its own disadvantages, too i.e. infection; shoot hyperhydricity and browning (Alp et al., 2010). This is why the plant materials were subjected to 2-stage surface sterilization. The initial disinfection stage was carried out with high concentration commercial bleach to remove soil-based infection/contamination sources. After the 1 st stage, the plant materials were trimmed down to make them ready for 2 nd stage disinfection process in order to prevent further infection/contamination. Since the results obtained from present study are highly positive regarding disinfection, 2-stage surface sterilization can be recommended for future studies.

Evaluations of media combinations
The micropropagation was conducted with two media combinations until 3 rd subculture. In previous studies, it was stated that MS basic medium supports in vitro shoot formation in globe artichoke (De Leo and Greco, 1973;Ancora et al., 1981;Pécaut et al., 1983;Iapichino, 1996;Alp et al., 2010;El-Zeiny et al., 2013;López-Pérez and Martínez, 2015;Dawa et al., 2018). Throughout the experiments, media combinations were carried out with MS basic medium and it was found that MS basic media supplemented with 0.05 mg L -1 BA + 0.005 mg L -1 IBA combination (No.1) was more successful than media combination of 0.25 mg L -1 BA + 0.025 mg L -1 IBA (No.2) in terms of general appearance, growth and development of plantlets. It is why all the plantlets originating from media combination No.2 were discarded and plantlets coming from media combination No.1 were used for the rest of the experiment period. Results on media evaluation clearly showed that medium No.1 was superior over medium No.2. In other words, lower concentrations of BA and IBA resulted with good growth and development of in vitro plantlets of the all globe artichoke cultivars used. Similar findings were also reported in previous studies. In a study conducted by Bedini et al. (2012), they reported that combination of BA and IBA at reduced concentrations was very successful for in vitro propagation of globe artichoke. Several studies also reported that the use of low plant growth regulators concentrations promoted and enhanced the plantlet quality (Ancora et al., 1981;Pécaut et al., 1983;Rossi and De Paoli, 1992;Morone Fortunato and Ruta, 2003;Castiglione et al., 2007;Bedini et al., 2012). It is assumed that increasing plant growth regulator concentration stimulate negative symptoms such as browning and shoot hyperhydricity (Rossi and De Paoli, 1992;Brutti et al., 2000;Pacifici et al., 2007;Bedini et al., 2012). So, findings of the present study are in accordance with previous studies.

Multiplication rates (MR)
There was no rapid increase in MR at the beginning of culture for OP and F1 hybrid globe artichoke cultivars. As the number of subcultures increased, the shoots became well adapted to in vitro conditions and MR, therefore, increased (Table 1). Many researchers think that low MR at the beginning of culture is associated with the initial stress of shoots and as a result of getting adapted to in vitro conditions in due time either MR increases or inter subcultural fluctuations are minimized (Phillips et al., 1994;Zucchi et al., 2002;Rey et al., 2013). Findings of present study about MR are, thus, in agreement with previous studies. MR showed variation based on cultivar being OP or F1 hybrid and as well as number of subcultures. 'Bayrampaşa' OP globe artichoke cultivar and 'Madrigal' and 'Imparator' F1 hybrid globe artichoke cultivars reached the maximum MR at the 10 th subculture. On the other hand, 'Green Globe' and 'Sambo' F1 hybrid globe artichoke cultivars were reached to maximum MR at the 8 th subculture, while 'Sakız' OP globe artichoke cultivar and 'Olympus' F1 hybrid globe artichoke cultivar reached to maximum MR at the 6 th subculture. Although the 'Bayrampaşa' OP and 'Imparator' F1 hybrid globe artichoke cultivars reached the maximum MR in the 10 th subculture, these values were found to be the highest MR values when the overall study was evaluated. Although, Madrigal F1 hybrid cultivar reached the maximum MR in the 10 th subculture like these cultivars, the MR of 'Bayrampaşa OP and 'Imparator' F1 hybrid globe artichoke cultivars were about 5 times higher than 'Madrigal' F1 hybrid cultivar. Although, 'Sakız' OP cultivar reached the maximum MR in the 6 th subculture there were fluctuations between the 7 th and 10 th subcultures. The most fluctuations on MR values were recorded in F1 hybrid cultivars, 'Madrigal', 'Sambo' and 'Green Globe'. In previous studies, it was revealed that there was a relationship between the number of subcultures and in vitro proliferation. El-Zeiny et al. (2013) reported that they recorded an increase in in vitro production until the 5 th subculture in a total of 7 subcultures conducted in their study. Similarly, Dawa et al. (2018) stated that when the number of subcultures was increased in vitro multiplication rate also increased until a certain number of subcultures. It is thought that decision of how many subcultures needed to reach to certain number of plants should be done heavily depends on the cultivars used for in vitro micropropagation.

Axillary shoots formation
There was no statistically significant difference, in terms of average axillary shoots formation, among cultivars regardless of being OP or F1. On the other hand, there were statistically significant differences among all cultivars regarding axillary shoots formation based on number of subculture (Table 2). Among all evaluated globe artichoke cultivars, 'Imparator' F1 hybrid had with 12.33 axillary shoots/explant at 7 th subculture. Considering the response of OP and F1 hybrid cultivars to subculture averages, the OP cultivars demonstrated consistent values between each other with respect to axillary shoot formation in different subcultures, while F1 hybrid cultivars had fluctuations on axillary shoot formation. 'Green Globe' F1 demonstrated the lowest axillary shoot formation at the 10 th subculture due to heavy infection problem. Each cultivar belonging to OP or F1 hybrids demonstrated variation in itself. It is clearly demonstrated that applying a certain number of subcultures is a necessity to obtain certain number of in vitro plants, regardless of being OP or F1 hybrid cultivar. Bedini et al. (2012) stated that maximum shoot proliferation was obtained after 3 rd subculture. Similar reports were also reported by Pacifici et al. (2007). Findings of present and previous studies clearly indicated that globe artichoke's proliferative process and number of needed subcultures depends on cultivars.

Leaf formation
When the average number of leaves formed on the explant of the globe artichoke cultivars was evaluated, it was seen that OP cultivars had more positive results; especially 'Sakız' formed the maximum average with 20.8 leaves/explant. When leaf formation considered, the OP cultivars reached to maximum leaf formation/explant at the 5 th subculture. It was clearly seen that each F1 hybrid cultivar predominantly reached to maximum leaf formation/explant at the 4 th subculture although two of them reached at 5 th and 8 th subcultures. According to Pacifici et al. (2007), the increase in the number of leaves formed in in vitro conditions will cause more water loss and cause difficulties in the adaptation of artichoke plants to in vivo conditions. Many previous studies demonstrated that leaves of in vitro plants have different morphological structures in a comparison to in vivo leaves. Accordingly, since they have different leaf morphology, in vitro plants need to adapt themselves to unfavorable environmental conditions of in vivo by changes on morphological, anatomical, and physiological features (Brutti et al., 2000;Cavallaro et al., 2007).

Evaluation of in vitro rooting, acclimatization and survival percentages of cultivars
In this study, the rooting success of the plantlets after micropropagation stage was also evaluated (Table  4). To achieve well-formed root formation several media combinations were tested. Among rooting media combinations ½ MS medium combined with 10.0 mg L -1 IAA + 1.0 g L -1 activated charcoal was determined to have positive results in terms of in vitro rooting. Although some of the plantlets all of cultivars obtained between 6 th and 9 th subcultures in the medium used for micropropagation were rooted, their rooting percentages were not enough, so they were not taken into further consideration. Regarding in vitro rooting percentage, 'Sakız' OP cultivar was the best, while 'Sambo' F1 hybrid cultivar had the lowest rooting. At the acclimatization stage it was obvious that F1 hybrid globe artichoke cultivars adapted to in vivo conditions better than OP globe artichoke cultivars. Obtained results clearly revealed that right media combination is a necessity for well root formation and right media combination may vary based on genotype. Similar findings were also reported by Cavallaro et al. (2004); Iapichino (2013); López-Pérez and Martínez (2015). As stated in previous studies, the most challenging part of in vitro micropropagation studies for globe artichoke is the in vitro rooting stage (López-Pérez and Martínez, 2015;Ercan, 2016). As seen in present study, in vitro rooting rate is quite low due to genotype-related differences. This is a limiting step for the micropropagation, suggesting that various optimization studies are still needed for in vitro rooting (López-Pérez and Martínez, 2015).
In the present study, the acclimatized plant percentages were found to be rather low. In a previous study conducted by Cavallaro et al. (2007), it was found that there was a relationship between acclimatization time and acclimatized plants. Researchers revealed that the most favorable percentage of acclimatized plants was obtained in the September plantation time due to thermal conditions. On the other hand, in the current study, the plantation was performed in March. Thus, it is thought that having low level of the acclimatized plant percentages can be attributed to unfavorable thermal conditions in March.

Conclusions
The present study was aimed to comparatively evaluate in vitro micropropagation efficiency of 2 important local OP globe artichoke cultivars ('Bayrampaşa', 'Sakız') and 5 F1 hybrid globe artichoke cultivars ('Olympus', 'Madrigal', 'Sambo', 'Green Globe' and 'Imparator'). The experimental results clearly revealed that OP cultivars and F1 hybrid cultivars reacted differently to in vitro mass micropropagation. Besides genotypic differences, it was also observed the number of subcultures was one of the essential factors that affect in vitro multiplication rates in both OP and F1 hybrids. Although the F1 hybrid cultivars were ahead of the OP cultivars at the acclimation stage, the multiplication rates and rooting values of the OPs were found almost similar to F1 hybrid cultivars. Therefore, if there are local OP cultivars well adapted to certain ecological conditions as well as having good yield and fruit quality, they can be used in in vitro mass propagation as an alternative to F1 hybrid cultivars which are expensive and sometimes not easy to find.

Authors' Contributions
Both authors read and approved the final manuscript.