2025 Volume 90 Issue 3 Pages 153-158
Tripleurospermum hygrophilum is a threatened rare endemic species in Türkiye. In this work, an effective in vitro propagation protocol was developed for T. hygrophilum using in vitro techniques. Nodal explants were used to test in vitro shoot induction in Murashige–Skoog (MS) medium supplemented with several combinations of cytokinins and auxins. The highest number of shoots (2.90±0.55) among all the media was obtained from MS medium supplemented 0.25 mg L−1 6-Benzylaminopurine+0.1 mg L−1 Indol-3-butyric acid. Rooting was observed in all auxin concentrations studied, while the highest number of roots (15.85±2.87) was found in MS medium containing 0.25 mg L−1 1-Naphthaleneacetic acid (NAA). The survival rate of the plants transferred to the external environment was found to be 98%. No changes in chromosome number (2n=2x=18) and genome size between regenerants and mother plants in natural population were detected. This in vitro propagation protocol developed herein can be used as conservation activites of the threatened endemic species of T. hygrophilum.
In vitro propagation with a small plant tissue allows the short-term conservation of threatened plants (Liao et al. 2006; Inceer et al. 2022). The development of in vitro plant collections is considered a method of conserving native flora plants as well as an effective method for conserving biodiversity. During the plant tissue culture process, micropropagated plants undergo plant growth processes that result in cytological and chromosome abnormalities. This may result in genetic instabilities during the production of regenerants. These genetic instabilities, known as somaclonal variations, can be caused by the genotype of the explant, the culture regime, and plant growth regulators (Bairu et al. 2011).
Determination of genetic stability of plants propagated in vitro can be determined using morphological, cytological, biochemical and molecular techniques (Benson 1999; Hawkes et al. 2000). The most common genetic instability in in vitro cultures is changes in ploidy level. The basic and reliable method for determining ploidy level is to count chromosomes in metaphase of mitosis (Neumann et al. 2009). In many studies, chromosome counting is used to control the genetic stability of plants propagated in vitro (Godo et al. 1998; Mallón et al. 2010; Clarindo et al. 2012; Inceer et al. 2022). Flow cytometric (FCM) analysis is another common method used to determine genetic stability in plants grown in vitro (Mallón et al. 2010; Clarindo et al. 2012; Ulvrova et al. 2021). Flow cytometry, which uses nucleic acid-specific fluorescent dyes, is a rapid, simple and reproducible technique for the evaluation of DNA content and ploidy variations in plant tissue cultures (Escobedo-Gracia-Medrano et al. 2018).
Tripleurospermum hygrophilum (Bornm.) Bornm., which is a member of the family Asteraceae, is a rare endemic species in Türkiye. The native range of this species is norhwest and west of Türkiye. It grows in the moist meadows of the subalpine mountains at an altitude of 900 m. (Enayet Hossain 1975). Conservation status of T. hygrophilum is endangered (EN) included in the IUCN Red List categories (Ekim et al. 2000). Besides, its natural populations is under several antropogenic pressures such as tourism, fire and fragmentation. Hence, conservation activities are necessary as soon as possible. No studies have been conducted so far to conserve the T. hygrophilum.
The aim of the present study is to develop an effective in vitro propagation protocol for T. hygrophilum, and to test the genetic stability of propageted plants using chromosome counting and genome size.
The ripe achenes obtained from the collection of T. hygrophilum (Türkiye: Izmir, Yamanlar Mountain, 900 m, 24.05.2011, Inceer 810, KTUB) were used for tissue culture. The achenes were washed in tap water for 30 min and then were sterilized with 70% (v/v) ethanol (EtOH) solution for 30 s. After removing the ethanol with deionized sterile water, the achenes were surface sterilized with 3% (v/v) sodium hypochlorite (NaOCl) solution for 10 min. Sterilized achenes were washed with deionized sterile water 3 times for 15 min and cultured on 30 mL plant growth regulators-free Murashige and Skoog (MS) (Murashige and Skoog 1962) (Duchefa Biochemie) basic medium in a culture vessel 98.5×59 mm (Sigma-Aldrich). The pH values of the medium were adjusted to 5.8 with 1 M HCl or 1 M NaOH before autoclaving. Achenes were incubated in a growth chamber under a condition of 16-h light and 8-h dark cycle at 25±1°C with a light intensity of 50 µmol m−2 s−1. Twenty-eight-day-old seedlings were used as a source of explants.
Shoot regenerationNodal segments obtained from seedling shoots were cultured on MS basal medium supplemented with different concentrations of plant growth regulators (PGRs) (6-benzylaminopurine-6-BA, kinetin-KIN, 6-(y,ydimethylallylamino)-purine-2iP, indol-3-butyric acid-IBA, 1-naphthaleneacetic acid-NAA (Table 1) as well as vitamins, 2% (w/v) sucrose and 0.8% (w/v) phyto agar. The PGRs-free MS was used as control. Each treatment contained a total of 20 healthy shoots and each experiment were carried out in triplicate.
| Control | Cytokinin (mg L−1) | Auxin (mg L−1) | Shoot number | Shoot length (mm) | Node number | |||
|---|---|---|---|---|---|---|---|---|
| KIN | 6-BA | 2iP | IBA | NAA | ||||
| 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 2.00±0.56de | 36.34±3.20hi | 3.10±0.72e |
| 0.25 | 0.1 | 1.95±0.51cde | 34.87±3.15gh | 2.80±0.70de | ||||
| 0.25 | 0.1 | 2.90±0.55g | 38.70±2.91jk | 3.90±0.85gh | ||||
| 0.25 | 0.1 | 1.20±0.41a | 28.21±1.84ab | 1.90±0.55ab | ||||
| 0.25 | 0.1 | 1.85±0.59 cd | 35.96±3.09hi | 3.60±0.68fg | ||||
| 0.25 | 0.1 | 1.90±0.55 cd | 33.88±2.57fg | 3.05±0.76de | ||||
| 0.25 | 0.1 | 1.10±0.31a | 28.62±2.22bc | 1.75±0.44a | ||||
| 0.5 | 0.1 | 2.25±0.55ef | 37.37±3.11ij | 4.20±0.77h | ||||
| 0.5 | 0.1 | 1.95±0.60cde | 39.43±2.15k | 3.10±0.64e | ||||
| 0.5 | 0.1 | 1.10±0.31a | 32.19±2.31def | 2.05±0.69ab | ||||
| 0.5 | 0.1 | 1.20±0.41a | 31.93±1.62de | 2.25±0.64bc | ||||
| 0.5 | 0.1 | 2.35±0.49f | 38.67±1.91jk | 3.25±0.72ef | ||||
| 0.5 | 0.1 | 1.65±0.49bc | 33.53±2.50efg | 2.85±0.75de | ||||
| 1.0 | 0.1 | 1.35±0.49ab | 30.03±2.86c | 2.30±0.66bc | ||||
| 1.0 | 0.1 | 1.25±0.44a | 31.76±2.68d | 2.15±0.59abc | ||||
| 1.0 | 0.1 | 1.15±0.37a | 31.78±2.36d | 2.85±0.75de | ||||
| 1.0 | 0.1 | 1.85±0.49 cd | 29.51±2.13bc | 2.60±0.68 cd | ||||
| 1.0 | 0.1 | 1.30±0.47a | 26.88±1.91a | 2.10±0.64ab | ||||
| 1.0 | 0.1 | 1.25±0.44a | 33.89±2.52fg | 2.00±0.65ab | ||||
The results represent three replicates of 20 plants per MS treatment for shoot proliferation after 4 weeks of culture. According to the Duncan’s multiple range test at p=0.05, different letters in a column represent significant differences.
MS medium strengthened with different concentrations of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) was used for rooting (Table 2). The plant growth regulators-free MS was used as control. Each treatment contained a total of 20 healthy shoots and each experiment were carried out in triplicate. Rooted shoots were transferred to vermiculite medium containing Hoagland nutrient solution adjusted to pH 5.8 in 71×71 mm transparent light-transmitting plastic containers. The plants were acclimatized by gradually decreasing the humidity in the same culture room conditions. After that, the surviving plants were taken into a mixture of peat and forest soil at a ratio of 1 : 1 (v/v) and transferred to the botanical garden. The growth and development of the plants, which were transferred to the botanical garden and completed their acclimatization, were followed during one year.
| Control | IBA (mg L−1) | NAA (mg L−1) | Root number | Root length (mm) | Secondary root number |
|---|---|---|---|---|---|
| 0.0 | 0.0 | 0.0 | 11.35±1.76ab | 45.65±2.61d | 12.25±2.55d |
| 0.25 | 15.30±2.27ef | 47.64±2.63e | 15.85±1.81e | ||
| 0.5 | 10.20±1.85a | 29.48±2.06c | 11.75±2.73 cd | ||
| 1.0 | 14.00±2.38de | 24.31±2.43ab | 10.70±1.59c | ||
| 0.25 | 15.85±2.87f | 50.89±2.89f | 14.40±2.70e | ||
| 0.5 | 13.10±2.38 cd | 25.57±2.06b | 8.50±2.76b | ||
| 1.0 | 11.85±2.58bc | 23.49±3.14a | 6.45±1.85a |
The results represent three replicates of 20 plants per MS treatment for rooting after 4 weeks of culture. According to the Duncan’s multiple range test at p=0.05, different letters in a column represent significant differences.
For chromosome counting, the root tips rooted plants were pretreated in 2 mM 8-hydroxyquinoline solution at 18°C for 6 h. They were fixed with a 3 : 1 mixture of 96% ethanol–acetic acid at 4°C for 24 h and hydrolyzed at 60°C in 1 M HCl for 10–12 min. After the root tips taken from hydrolysis were dyed with Shiff-Reagent for 2–3 h, kept at room temperature, squashes were made in 45% acetic acid (Inceer and Beyazoglu 2004). Five well-spread metaphase plates from five plantlets were used for chromosome counting.
Flow cytometric analysisThe young leaves randomly selected from in vitro propagated plants belonging to three individuals in botanical garden were used for FCM analysis (Inceer et al. 2022). The leaf fragments of the sample plant and the standard plant (Zea mays L., 2C=5.48 pg) were chopped using a razor blade in 1 mL of woody plant buffer and transferred to tubes after being filtered through a 30 µM nylon filter (Loureiro et al. 2007). 50 µg/mL RNase and 50 µg/mL propodium iodide (PI) were added to the obtained nucleus suspension and incubated on ice in a light-free environment until measurement. Samples were then analyzed using a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) and histograms were generated after analyses of at least 10,000 nuclei per sample (Inceer et al. 2022). Nuclear DNA content (2C) was then calculated from mean values of G1 peaks (Inceer and Aksu Kalmuk 2020)
Nodal cuttings obtained from germinating achenes were used in shoot propagation. The highest shoot number (2.90±0.55) was recorded on MS medium strengthened with 0.25 mg L−1 6-BA+0.1 mg L−1 IBA (Table 1; Fig. 1A). Significant differences between 6-BA, control, and other tested cytokinins in terms of shoot number were determined. Besides, the lowest response in terms of shoot number was obtained on shoots in media containing 2iP (Table 1). The results showed that the highest shoot length (39.43±2.15) was obtained from MS medium strengthened with 0.5 mg L−1 6-BA+0.1 mg L−1 IBA among all treatments and the lowest shoot length (26.88±1.91) was found on MS medium containing 1.0 mg L−1 6-BA+0.1 mg L−1 NAA (Table 1).
(A) Shoot multipication on MS suplemented with 0.25 mg L−1 6-BA+0.1 mg L−1 IBA. (B) Rooting on MS medium containing 0.25 mg L−1 NAA after four weeks. (C) The flowering in the botanical garden after acclimatization. (D) Fruting stage. (E) Ripe achenes obtained from T. hygrophilum propagated in vitro grown in botanical garden. Scale bars=2 cm (A, B, D and E) and 3 cm (C).
In this study, 100% of the shoots were rooted. The media containing low concentrations of IBA and NAA were found to be more effective than other rooting media in terms of root formation. While the first rooting was observed on the MS medium strengthened with 0.25 mg L−1 NAA at the end of the first week, it was noted that all shoots were rooted at the end of the fourth week. The highest mean root number (15.85±2.87) was recorded on the MS media strengthened with 0.25 mg L−1 NAA (Fig. 1B), followed by the MS media containing 0.25 mg L−1 IBA (Table 2). With regard to the other root parameters, the highest root length (50.89±2.89) was observed on the MS medium strengthened with 0.25 mg L−1 NAA, this medium was found significantly different from all the rooting mediums (Table 2). Among the all rooting media, the highest number of secondary roots (15.85±1.81) was obtained from MS medium supplemented with 0.25 mg L−1 IBA. Differently, the highest mean secondary root number (15.85±1.81) was obtained from MS medium strengthened with 0.25 mg L−1 IBA (Table 2).
AcclimatizationIn the botanical garden, the plants continued to develop and it was determined that the plants adapted to this environment with 98% success (Fig. 1C, 1D). Morever, it was observed that the plants, which successfully adapted to the botanical garden and continued their development, started to bloom in the second year (Fig. 1C). First flowering took place 350 days after it was transferred to the external environment and on top of all that the plants that have completed the flowering phase have formed achene (Fig. 1D, 1E).
Genetic analysesAccording to the results of chromosome counting revealed that the chromosomes number in each experimental group were 2n=2x=18 (Fig. 2A, 2C–E). Likewise, according to results of flow cytomertric analysis, the nuclear DNA content (2C-value) of propagated plants was 4.88±0.47 pg (Fig. 2B), and the monoploid genome size (1C-value) was 2.44 pg.
(A) T. hygrophilum chromosomes at metaphase in rooting medium (1 mg L−1 IBA+MS). (B) Flow cytometric histogram of relative DNA content of micropropagated plants. (C) T. hygrophilum chromosomes at metaphase in PGRs-free MS medium (Control). (D) T. hygrophilum chromosomes at metaphase in rooting medium (0.25 mg L−1 NAA+MS). (E) T. hygrophylum chromosomes at metaphase in rooting medium (0.25 mg L−1 IBA+MS). Scale bars=5 µm (A, C–E).
The tissue culture, which is one of the biotechnological methods; it has become more and more important as a helpful technique for the conservation activies of threatened plant species (Benson 1999). In the present study, in vitro propagation protocol was developed for threatened endemic species T. hygrophilum, and the genetic stability of propagated plants was tested with chromosome counting and flow cytometric analysis. The plants obtained in vitro via direct shoot regeneration have well grown and developed including flowering and fruiting periods in the field conditions. The plants cultivated in botanical garden have been similar morphological characteristics such as habit and achene (Enayet Hossain 1975; Inceer et al. 2012) as well as genetical traits with in vivo mother plants Thus, ex situ collections of this species were succesfully achieved.
There are studies on Asteraceae members with tissue culture techniques, but studies in Tripleurospermum have been limited to only four endemic species (namely, T. insularum (Cuce et al. 2022), T. fissurale (Inceer et al. 2022), T. ziganaense (Cuce and Inceer 2024) and T. baytopianum (Cuce et al. 2025). Since there is a lack of micropropagation protocol, we have attempted to establish an efficient micropropagation system for rare endemic T. hygrophilum.
In micropropagation studies, the balance between the use of auxin and cytokinin is important for effective propagation (Pollard and Walker 1990). It is known that the use of low concentrations of auxin together with cytokinins on in vitro shoot production of the same family members is effective in shoot production (Trejgell et al. 2018; Talla et al. 2019; Nithya and Kamalam 2021; Riahi et al. 2022). Within Tripleurospermum, it is known that there is no changes in ploidy level in propagated plants from the combinations in low concentrations of cytokinins. Our findings showed that the combination of low concentrations of auxin with a cytokinin are necessary for effective organogenesis of T. hygrophilum. Similar results are reported for other species of Tripleurospermum, such as T. fissurale, T. insularum and (Cuce et al. 2022; Inceer et al. 2022; Cuce and Inceer 2024).
It has already been reported in many studies that 6-BA is the most effective plant growth regulator on shoot formation for many members of the Asteraceae family, and it is also combined with IBA to promote shoot formation (Grigoriadou et al. 2011; Blando et al. 2021). In the present study, the highest shoot number and lenght in T. hygrophilum were obtained from MS medium containing 6-BA+IBA. Similarly, the the studies conducted by Cuce et al. (2022) in T. fissurale and Cuce and Inceer (2024) in T. ziganaense, the highest number of shoots was yielded from the same plant grow regulators, namely 6-BA+IBA.
In our study, the lowest shoot number was obtained from 2iP and NAA containing medium. Similar to our results, in the study in which the in vitro propagation protocol of Spilanthes acmella within Asteraceae family was established, it was found that MS medium containing 2iP+NAA was less effective than other mediums in terms of shoot parameters (Saritha et al. 2002). In addition, Cuce and Inceer (2024) reported that the 2iP+NAA combination was less effective in producing shoots than other combinations in the propagation of T. ziganaense.
The rooting stage is the phase in which the shoot-forming plants are rooted and prepared to be transferred from the in vitro to the ex vitro environments (Iliev et al. 2010). In many micropropagation studies with Asteraceae members, during the rooting phase, the highest root number was obtained from MS medium containing NAA (Rafiq et al. 2007; Corral et al. 2011; Okello et al. 2021). In the study by Inceer et al. (2022) in which T. insularum was micropropagated, the highest root number was obtained from MS medium strengthened with 0.5 mg L−1 NAA. Similarly, in the study conducted by Cuce et al. (2022) with T. fissurale, the highest root number was found on MS medium containing 0.5 mg L−1 NAA. In our work, in parallel with previous studies, it was noted that media containing NAA were more effective in root formation, and MS medium strengthened with 0.25 mg L−1 NAA was found to be more effective than other rooting media in increasing both root number and root length.
Chromosome counts and flow cytometric analysis are one of the basic methods to control of genetic stability of propagated plants. They are used alone or as a complementary method in many studies (Mallón et al. 2010; Clarindo et al. 2012; Ulvrova et al. 2021). Considering the in vitro protocol developed herein for T. hygrophilum, the propagated plants have the same chromosome number with mother plants (2n=2x=18, Inceer and Hayirlioglu-Ayaz 2010) in natural population. According to the results of flow cytometric analysis, there is no significant difference in genome size between propagated plants (1C=2.44) and mother plants (1C=2.48 pg, Inceer et al. 2018), as well. These findings show that the propagated plants are genetically stable.
The authors would like to thank Betül Ergin for technical supports, and TUBITAK (Project No 117Z588) for financial support.
Tugba Ergin: Investigation, Data curation, Visualization, Formal analysis, Methodology, Software, Writing – original draft, Writing – review & editing.
Huseyin Inceer: Conceptualization, Visualization, Investigation, Methodology, Writing – original draft, Writing – review & editing.
