In Vitro Propagation and Nuclear DNA Content of Tripleurospermum Insularum (Asteraceae) – A Critically Endangered Insular Endemic Species From Turkey

Tripleurospermum insularum Inceer & Hayirlioglu-Ayaz (Asteraceae) is a critically endangered endemic species in Turkey that is face the risk of extinction as a result of the fragmentation of its habitat as well as overgrazing and trampling of its natural population. However, the protocol for micropropagation of this threatened species has not been developed yet. Here, its regeneration ability on MS media supplemented with different plant growth regulators were evaluated using nodal segments. The higher number and length of shoot per explant was achieved with the addition 4.6 µM ZEA and 0.5 µM IAA to the culture medium. Besides, the highest node number of shoot per explant was obtained from MS medium supplemented with 4.6 µM ZEA and 0.5 µM IBA. Flow cytometric analysis also revealed that most of the in vitro developed shoots of T. insularum possessed similar nuclear DNA content as well as ploidy level as initial material and plants from natural population. In vitro rooting of shoots was achieved at 100 % eciency containing 2.9 µM IAA. Rooted and well-developed plantlets were initially acclimatized under greenhouse conditions and then moved to the botanical garden, where they matured and owered. Finally, 76% and 74% survivals were achieved during the acclimatization process, respectively. This is the rst report of a successfully developed micropropagation protocol of threatened T. insularum for its ex situ conservation. present results indicate that the rate of in vitro achene germination in T. insularum were relatively low because of recalcitrant, which is in line with our previous ex vitro germination test for chromosome counting (Inceer and Hayırlıoglu-Ayaz 2014, data not Leyva-Peralta et al. (2019) noted that the low seed germination yields have attributed to the heterogeneous maturation of the seeds in the Asteraceae, resulting in a high proportion of empty seeds. According to Benson (2000b) and Leyva-Peralta et al. (2019), recalcitrance the inability of plant cells, tissues, and organs to respond to tissue culture. However, the obtained seedlings of T. insularum IAA NAA effective than IBA in the induction of roots in regenerated shoots of T. insularum. root This study is the rst report of the ecient in vitro propagation of threatened insular endemic T. insularum. Nodal explants were used as excellent starting material for axillary shoot multiplication. Multiple shoots possessed almost the same nuclear DNA content as native plants, except when they were grown on medium supplemented with KIN plus IBA, ZEA plus IBA, KIN plus NAA, 6-BA plus NAA, and 2iP plus NAA. Additionally, the present results conrm that the proposed protocol guarantees in vitro production of T. insularum with genetic stability. The plants obtained in vitro via direct organogenesis exhibited normal development, were fertile, and seemed to adapt well to the botanical garden. Hence, this protocol may be used in future conservation programs such as in situ activities on population restoration and cryopreservation of this species as well as other threatened endemic species of Tripleurospermum.


Introduction
The overall loss of biodiversity as well as the number of threatened plant species in the world has increased in recent years due to several anthropogenic pressures and/or environmental factors (Corral et al. 2011). Therefore, it is imperative to implement conservation strategies for the conservation and propagation of phytogenetic resources from each geographic region ( The current International Union for Conservation of Nature (IUCN) Red List categories and criteria are constructed to assess the threatened status of species or lower taxa on global or sub-global (national or regional) levels as a means of classifying the relative risk of extinction of taxon (Inceer and Aksu Kalmuk 2019). Additionally, IUCN Red List database shows that plant extinctions are dominated by insular species, and the extinction data are associated with lower competitive ability and anthropogenic factors (Gray 2018). Indeed, insular species are exposed to multiple threats, such as invasions, land use change, and global climate change Ex situ conservation methods have played an important role in the conservation of threatened plants (Coste et al. 2012). In this context, in vitro culture techniques have permitted an increasingly important viable system for rapid, true-to-type mass multiplication as well as conservation of threatened endemic and rare species (Piovan et  According to Inceer and Hayırlıoglu-Ayaz (2014), T. insularum is classi ed within CR category of IUCN. The Red List extinction data of this species are associated with lower competitive ability and anthropogenic factors such as overgrazing, trampling and fragmentation. The population is therefore facing extinction in the near future, unless in situ and/or ex situ conservation strategies are adopted as soon as possible. To our knowledge, no investigations have been carried out on the in vitro culture of members of the genus Tripleurospermum. The aim of the present study is to develop the e cient in vitro micropropagation protocol for the threatened T. insularum and to evaluate the genetic delity of regenerants in order to ex situ conservation of this insular endemic species.

Plant material and achene germination
The mature achenes of T. insularum intended for evaluation as explants were supplied from the seed collection of Dr. (Sigma). At the end of the 30 th day, the plantlets were evaluated for germination percentage to determine which of these two main basal media was better suited to T. insularum.

Shoot proliferation
In the shoot multiplication studies, nodal segments obtained from seedling shoots after the third subculture were used as an explant.
MS basal medium including vitamins and containing 2% (w/v) sucrose (Duchefa) and 0.8% (w/v) phyto agar (Duchefa) was selected as the most suitable medium for shoot proliferation studies. 6-benzylaminopurine (6-BA, 4.4 µM), KIN (4.7 µM), 6-(y,ydimethylallylamino)-purine (2iP, 4.9 µM), thidiazuron (TDZ, 4.5 µM) and zeatin (ZEA, 4.6 µM) in combination with indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA) (0.5 µM) were added to the medium to support the basic basal medium and increase the shoot multiplication rate. All plant growth regulators (PGRs) used in this study and supplied by Sigma were lter-sterilized with 0.22-µm lters, except for 6-BA and NAA, and were added to the cooled media after autoclaving. The pH of the media was adjusted to 5.8 with 1 M HCl or 1 M NaOH before autoclaving. All cultures were maintained at 24 ± 2°C under a 16/8 h photoperiod at a photosynthetic ux of 50 μmol m −2 s −1 , provided by cool daylight uorescent lamps. The subculturing protocol was performed every four weeks. Multiplication rates were calculated by assessing the number of shoots per explant, length of shoots, the number of leaves on each shoot, callus formation, and plant quality (internode length and shoot thickness).

Nuclear DNA content
Flow cytometry was used to check for the stability of nuclear DNA content as well as ploidy level in in vitro derived shoots of T. insularum. The leaves of 30-day-old seedlings and of shoots multiplied on MS media supplemented with cytokinins (KIN, 2iP, TDZ, 6-BA and ZEA) and auxins (IBA, IAA, and NAA) were used for ow cytometric analysis. Leaf fragments of the sample plant and the standard plant (Zea mays L.) were chopped up using a razor blade in 1 mL of woody plant buffer (Loureiro et al. 2007, 0.2 M Tris HCl, 4 mM MgCl 2 .6H 2 O, 2 mM EDTA, Na 2 .2H 2 O, 86 mM NaCl, 10 mM K 2 S 2 O 5 , 1% PVP-10, 1% (v/v) Triton X-100, pH 7.5), supplemented with 50 μg mL -1 propidium iodide and 50 μg mL -1 DNase-free RNase, ltered through a 30-μm mesh and stored on ice, in the dark, until measurement. Three independent samples were extracted, ltered and then measured using a BD Accuri™ C6 instrument. Usually 10.000 nuclei per sample were analysed for nuclear DNA content and absolute values (Inceer et al. 2016). DNA content was then calculated from mean values of G1 peaks according to the following formula: Root induction MS media, each individually supplemented with 2.5 µM IBA, 2.9 µM, IAA and 2.7 µM NAA and without growth regulators, were again selected for rooting the well-proliferated and su ciently elongated shoots (≥ 20 mm). Four weeks after the transfer of shoots to the root media, the rooting percentage, number of roots per shoot, root length and secondary root number (lateral roots on the longest root) were calculated to evaluate the rooting success. Each experiment was carried out in triplicate.

Acclimatization
After rooting studies, rooted and well-developed plants were selected to determine their survival rates under greenhouse conditions as well as in the botanical garden. The greenhouse humidity and temperature were adjusted to 80-85% and 26 ± 2°C, respectively. After 30 days, the plants were moved to a eld in the botanical garden. While 100% peat was employed in the greenhouse, 1:1 (v/v) peat and forest soil was used for transferring the plantlets to the botanical garden.

Statistical analysis
For all germination experiments, ve achenes were placed into each Magenta vessel, and four vessels were prepared per treatment. Each treatment was performed in triplicate. Statistical differences between obtained from shoot proliferation, root induction and ow cytometric studies were calculated on Statistical Package for the Social Sciences software (SPSS version 21). Duncan's multiple range test (DMRT, 95% con dence level) from one-way analysis of variance (ANOVA) was used to detect the statistical signi cance of differences among the mean values in shoot multiplication and root induction. Nuclear DNA data were evaluated using ANOVA and Dunnett's test at P = 0.05.

In vitro germination
The rst germination data were obtained at the end of days 8 and 10 after inoculation of the achenes in MS and B5 media, respectively. The MS medium had a greater effect than B5 medium, and yielded 8% more achene germination. Fifty-eight percent germination success was obtained in MS medium, compared to 50% in B5 medium. These two culture media exhibited signi cant differences in terms of numbers of germination achenes, (P < 0.05). MS medium was therefore employed in subsequent shoot multiplication studies of T. insularum.

Shoot proliferation
In micropropagation studies, the addition of PGRs to the basal media makes a signi cant contribution to growth development factors such as length of shoots, lateral shoot formation, and shoot thickness. Furthermore, these PGRs added as cytokinin and auxin combinations differ depending on the plant species and basal medium components. In vitro shoot proliferation from nodal explants was successfully achieved after 4 weeks. MS media supplemented with ZEA and IAA, ZEA and IBA or cytokinin and auxin-free media were found to be more effective in terms of all the tested parameters in shoot proliferation studies for T. insularum.
The maximum shoot number per explant (3.33 ± 0.48) for this species was recorded with MS medium supplemented with 4.6 µM ZEA and 0.5 µM IAA (Fig. 1a). Besides, TDZ applications gave lower values in terms of shoot numbers, and there were signi cant differences between these and other applications. MS medium with the same added cytokinin and auxin combination again reached the highest shoot elongation values with 39.68 ± 3.33 mm. Surprisingly, the cytokinin and auxin-free MS medium yielded the same shoot length value as MS medium with added 4.6 µM ZEA and 0.5 µM IAA (Table 1; Fig. 1a). On the other hand, MS medium containing 6-BA combined with any auxin was found to be unsuitable in terms of shoot length among all the applied cytokinin and auxin combinations. The lowest shoot length (13.48 ± 1.32) for this species was obtained from MS media containing 4.4 µM 6-BA and 0.5 IAA µM (Table 1). Signi cant differences were also observed between ZEA as well as control application and other applied cytokinins in terms of shoot length (P < 0.05).
In contrast to the mean values for shoot number and shoot length, MS medium supplemented with 4.6 µM ZEA and 0.5 µM IBA yielded the highest node number value with 10.67 ± 0.76 nodes per explant. An interesting result for this parameter was also obtained from control with 10.46 ± 1.06 nodes per explant (Table 1). On the other hand, there were signi cant differences between these two applications and the others (P < 0.05).
In the present study, evaluation of callus formation has revealed a signi cant response in all applications except for control and KIN plus IAA applications. Among all the treatments, TDZ in combination with IBA or IAA were more effective than other treatments in callus formation. TDZ in combination with IBA or IAA alone resulted in 100% callus formation. Although ZEA and NAA (54.17%) and 2iP and IAA (50%) combinations also caused signi cant callus formation, statistically signi cant differences were observed between TDZ and these two applications (Table 1).

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The healthy shoots above 20 mm elongation were cultured in MS medium containing individually 2.5 µM IBA, 2.9 µM IAA, and 2.7 µM NAA, or auxin-free for root induction. The culture conditions during root initiation and root growth were the same as the shoot formation conditions. The obtained rooting successes differed according to the tested auxin type at the end of the 4 th week. Only IAA application yielded 100% rooting success for all tested plantlets. In contrast, the highest values in terms of the evaluated rooting parameters were obtained from auxin-free and NAA supported MS media.
Although there was little difference between the four tested different media in terms of root formation times, the rst root was seen on day 19 in MS medium supplemented with 2.7 µM NAA. Approximately 20 mm of root formation in culture conditions may be su cient for acclimatization studies (Fig. 1b). However, the seedlings were kept in culture medium for four weeks due to the delayed initial root induction and in order to keep culture times equal to shoot formation times. Depending on the rst root formation, the highest root numbers, as well as secondary root numbers, were obtained from this medium with 2.46 ± 0.48 and 17.63 ± 0.91, respectively ( Table 2). The differences between the highest and the lowest root number were shown in Table 2 (P < 0.05). An e cient root length was observed in auxin-free medium with 59.1 ± 3.91 mm, compared with other auxin-treated media (Fig. 2). The lowest mean root length with 46.33 ± 3.41 mm among the groups was observed in MS medium supplemented with 2.5 µM IBA. Signi cant differences were determined in terms of root length values between all the tested media for root induction (P < 0.05).

Genetic stability
The results obtained from ow cytometry analysis of propidium iodide-stained nuclei are summarized in Table 1. As shown in Table   1

Ex situ collection
The rooted and well-developed plantlets were initially acclimatized under greenhouse conditions. The plants were healthy, green and grew rapidly as well as some of them also formed buds and then bloomed under greenhouse conditions ( Fig. 1c and 1d). The survival frequency was approximately 76% for T. insularum under greenhouse conditions (Fig. 3). After 30 days, the plants were moved to a eld in the botanical garden. After a month, they developed new leaves, and some also formed buds and then bloomed ( Fig. 1e and   1f). The survival frequency was approximately 74% for T. insularum under botanical garden conditions. The micropropagated plants were phenotypically indistinguishable from their seed-derived counterparts.

Discussion
In vitro culture of plant tissues has been used in conservation programs for many threatened species and is especially important in small populations (Gonçalves et (Table 1). Among the various growth regulators tested, the best proliferation of shoots was obtained on the MS medium with 4.6 µM ZEA and 0.5 µM IAA combination (Table 1) In the present study, the rooting has occurred both with and without auxin in all tested media, but the presence of auxin greatly increased the rooting rate compared to auxin-free medium at the end of the 4 th week (  (Table 2).
On the contrary, auxin free MS medium yielded a better response for root length. The present results revealed that IAA and NAA were more effective than IBA in the induction of roots in regenerated shoots of T. insularum. Additionally, the incidence of root formation on auxin-free medium may be due to the presence of endogenous auxin in regenerated shoots.

Conclusion
This study is the rst report of the e cient in vitro propagation of threatened insular endemic T. insularum. Nodal explants were used as excellent starting material for axillary shoot multiplication. Multiple shoots possessed almost the same nuclear DNA content as native plants, except when they were grown on medium supplemented with KIN plus IBA, ZEA plus IBA, KIN plus NAA, 6-BA plus NAA, and 2iP plus NAA. Additionally, the present results con rm that the proposed protocol guarantees in vitro production of T. insularum with genetic stability. The plants obtained in vitro via direct organogenesis exhibited normal development, were fertile, and seemed to adapt well to the botanical garden. Hence, this protocol may be used in future conservation programs such as in situ activities on population restoration and cryopreservation of this species as well as other threatened endemic species of Tripleurospermum.  Tables   Table 1 Effects    The effects of different auxin types on in vitro rooting of T. insularum shoots Figure 3 Survival rates of plantlets transferred from different rooting media to the greenhouse conditions