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Jan 2, 2014 - Ex situ conservation of Dianthus giganteus d'Urv. subsp. banaticus (Heuff.) Tutin by in vitro culture and assessment of somaclonal variability by ...
Turkish Journal of Biology

Turk J Biol (2014) 38: 21-30 © TÜBİTAK doi:10.3906/biy-1303-20

http://journals.tubitak.gov.tr/biology/

Research Article

Ex situ conservation of Dianthus giganteus d’Urv. subsp. banaticus (Heuff.) Tutin by in vitro culture and assessment of somaclonal variability by molecular markers 1

2,

3

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1

Liliana JARDA , Anca BUTIUC-KEUL *, Maria HÖHN , Andrzej PEDRYC , Victoria CRISTEA 1 Al. Borza Botanical Garden, Babeş-Bolyai University, Cluj-Napoca, Romania 2 Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeş-Bolyai University, Cluj-Napoca, Romania 3 Department of Botany, Faculty of Horticultural Science, Corvinus University, Budapest, Hungary 4 Department of Genetics and Plant Breeding, Faculty of Horticultural Science, Corvinus University, Budapest, Hungary Received: 11.03.2013

Accepted: 26.07.2013

Published Online: 02.01.2014

Printed: 24.01.2014

Abstract: Ex situ conservation of Dianthus giganteus subsp. banaticus was obtained by in vitro culture on different culture media supplemented with phytohormones. Assessment of somaclonal variability over a period of preservation by in vitro culture is required prior to introduction and acclimatization in natural habitats. In this study we used 2 types of molecular markers, ISSR and multilocus SSR markers, which allow comparison of different alleles, to assess the somaclonal variability of endemic taxa Dianthus giganteus subsp. banaticus after a period of 24 months of preservation by in vitro culture. The assessment of somaclonal variability of D. giganteus subsp. banaticus shows some changes in the absence/presence of certain markers in some individuals. Key words: ISSR, SSR markers, Dianthus giganteus subsp. banaticus, ex situ conservation, in vitro culture, somaclonal variability

1. Introduction The efficient approach to plant conservation implies in situ and ex situ conservation, having as a main target the maintenance of appropriate genetic diversity of the concerned species. Ex situ conservation is a viable alternative for taxa that are highly endangered in their natural environment or may complement in situ conservation methods (Ramsay et al., 2000; Marriott and Sarasan, 2010; Johnson et al., 2012). In vitro multiplication and reintroduction into natural habitats of rare, endangered, or endemic plant species allows the preservation of these species and expands the natural population. On the other hand, maintenance of several genotypes in ex situ collections in different locations provides extra safety for the conservation of certain species. Traditional methods of storing genetic resources as seeds and plant collections have contributed to the conservation of the genetic resources of the world. Today, advanced, modern methods of plant biotechnology together with traditional in situ and ex situ conservation are applied on a larger scale for the conservation of plant genetic resources. Dianthus is one of the most diverse plant groups in Europe and is distributed throughout Eurasia and Africa (approx. 300 species); over 100 species of carnations occur in Europe, and more than 70 are endemic (Valente et al., 2010). The studied * Correspondence: [email protected]

taxa, Dianthus giganteus D’Urv. subsp. banaticus (Heuff.) Tutin, is endemic for the South-West Carpathians and is considered vulnerable in Romania by some botanists (Dihoru and Dihoru, 1994; Sârbu et al., 2003) and rare by others (Olteanu et al., 1994). Somaclonal variability, defined as variability induced by different variants of cells and tissue cultures (Bairu et al., 2011), may be induced by in vitro culture due to the phytohormones used and cytokinin:auxin ratio (hormonal balance), in vitro stress, culture duration, and other nutritional conditions (Devarumath et al., 2002). In vitro micropropagation as an asexuate process usually should not produce variability (Bairu et al., 2011), but in some instances somaclonal variability could appear. Regarding the conservation of certain endemic or rare taxa having low genetic variability in their populations, somatic variability could ensure a source of genetic variability and a greater possibility of acclimatization in a habitat. In order to develop a proper conservation program of endemic or endangered taxa, it is necessary to evaluate the genetic variability in the populations and between populations. After conservation it is important to investigate the genetic stability of plants in order to identify the somaclonal variation (Halmagyi and ButiucKeul, 2007). Molecular markers are valuable tools used

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in the analysis of genetic fidelity of in-vitro–propagated plants. Among the numerous molecular markers available, inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) are cost efficient and require lower amounts of DNA (Zietkiewicz et al., 1994). As the simple sequence repeats are abundant and scattered throughout the eukaryotic genomes, ISSR and SSR markers have been frequently used in establishing genetic stability of several micropropagated plants in horticultural crops such as wheat (Ateş Sönmezoğlu, 2012) and date palm (Hamza et al., 2012); trees such as Populus tremuloides (Rahman and Rajora, 2001) and Pinus pinea (Cuesta et al., 2010); and several endangered or rare species such as Zingiber rubens (Mohanty et al., 2011), Swertia chirayita (Joshi and Dhawan, 2007), and Dictyospermum ovalifolium (Chandrika et al., 2008). In this paper we report the in vitro multiplication and conservation of D. giganteus subsp. banaticus, assessment of genetic variability in their populations, and somaclonal variability in the regenerated plants using ISSR and SSR markers.

from in situ plants, 20 explants/individuals/sterilizing methods) was sterilized using several methods, as described by Jarda et al. (2011). Before sterilization the plant material was pre-sterilized by washing in water, rinsing in 5% Domestos, and a short rinse in 80% ethylic alcohol. Prelevation of plant explants was done from 3 populations from Romania, each with 3 individuals (Table 1). 2.2. In vitro culture After 30 days from initiation on D1 culture media representing MS basal medium (Murashige and Skoog, 1962) supplemented with vitamins, 20 g/L sucrose, 8.0 g/L agar, 1.0 mg/L 6-benzylaminopurine (BAP), and 1.0 mg/L alpha-naphthyl acetic acid (NAA), the explants were transferred to multiplication-stabilization medium (D2) containing MS basal medium with vitamins, 20 g/L sucrose, 8.0 g/L agar, and a hormonal balance favoring cytokinins (1.0 mg/L BAP and 0.1 mg/L NAA) for multiplication. The plants obtained by in vitro culture were used for optimum multiplication and rhizogenesis rate studies. We used different culture media: one with a hormonal balance favoring cytokinins for multiplication and favoring auxins for rhizogenesis and an MS medium supplemented with vitamins as control (Tables 2 and 3). For each variant of culture medium we used 16 uniondale explants/individuals.

2. Materials and methods 2.1. Plant material For the initiation of in vitro culture of D. giganteus subsp. banaticus taxa, plant material (nodal explants prelevated

Table 1. Populations of Dianthus giganteus subsp. banaticus used as plant material. Population

Individuals

Coordinates

1. Domogled Valea Cernei National Park

1.1, 1.2, 1.3

N: 44°52′36,2″; E: 22°26′12,9v

2. Porţile de Fier National Park

2.1, 2.2, 2.3

N: 44°38′51,4″; E: 22°17′17,3″

3. Cheile Ţesnei Gorges

3.1, 3.2, 3.3

N: 44°58′35,2″; E: 22°34′11,0v

Table 2. Variants of culture media used for multiplication. Variants

Components

M1

M2

M3

M4

M5

M6

M7

M8

M9

MS

MS

MS

MS

MS

MS

MS

MS

MS

NAA

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

-

K

0.1

1.0

-

-

-

-

-

-

-

BAP

-

-

0.1

1.0

-

-

-

-

-

2iP

-

-

-

-

10

15

-

-

-

TDZ

-

-

-

-

-

-

0.01

0.05

-

Sucrose (g/L)

20

20

20

20

20

20

20

20

20

Agar (g/L)

7.8

7.8

7.8

7.8

7.8

7.8

7.8

7.8

7.8

Macroelements, microelements, and vitamins

Hormones (mg/L)

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JARDA et al. / Turk J Biol Table 3. Variants of culture media used for rhizogenesis. Variants

Components

R1

R2

R3

R4

Macroelements, microelements, and vitamins MS

MS

MS

MS

IAA

-

-

-

1

NAA

-

0.1

1

-

K

-

0.5

0.5

0.5

Sucrose (g/L)

20

20

20

20

Agar (g/L)

7.8

7.8

7.8

7.8

Hormones (mg/L)

Cultures were grown at a constant 22 °C under a 16-h light photoperiod regime (cool-white fluorescent lights, 50 µmol s–1 m–2 photosynthetic photon flux density). Statistical analyses were done using ANOVA. Plant material used in molecular studies was represented by fresh leaflets of individuals (i) before the initiation of in vitro cultures (ii) after 24 months of in vitro culture. Genomic DNA was isolated from leaves using the CTAB method described by Doyle and Doyle (1987). Two types of molecular marker, ISSR and SSR, were used. 2.3. ISSR analysis ISSR amplifications were performed using the 6 pairs of primers (BC 809, BC 812, BC 826, BC 861, BC 873, and BC 878) used by Yilmaz et al. (2009) for analysis of genetic relatedness in the genus Prunus. Amplification was performed in a 0.2-mL tube containing: 500 mM of KCl, 15 mM of MgCl2, 10 mM of each dNTP, 1.25 µM of each primer, 5 U of Taq polymerase (Fermentas), and 25 ng of genomic DNA in a final volume of 12.5 µL. Amplification program: 1) T = 94 °C, 4 min; 2) T = 94 °C, 1 min; 3) primer annealing at 49 °C, 1.30 min; 4) elongation at 72 °C, 1.30 min; 5) final elongation at 72 °C, 7 min; steps 2–4 were repeated 40 times. Amplification was performed in a 2720 Thermal Cycler (Applied Biosystems). Amplicons were separated on 1% agarose gel stained with 0.5 µg/mL ethidium bromide and visualized under UV light. 2.4. SSR analysis Amplification was performed using 5 pairs of primers (MS-DCAMCRBSY, MS-DCDIA30, MS-DINCARACC, MS-DINGSTA, and MS-DINMADSBOX) developed by Smulders et al. (2000) for the genus Dianthus. PCR amplifications were performed in a 0.2-mL tube containing 2 mM of MgCl , 200 μM of each dNTP, 1 μM 2 of each primer, 1.5 U of Taq polymerase (Fermentas), and 25 ng of genomic DNA in a final volume of 25 μL using a Gradient Palm-Cycler (Corbett Life Science). Amplification program: 1) T = 94 °C, 4 min; 2) T = 94 °C, 50 s; 3) primer annealing at 56 °C, 50 s; 4) elongation T =

72 °C, 50 s; steps 2–4 were repeated 45 times. Amplicons were separated on 1.5% agarose gel, stained with 0.5 μg/ mL ethidium bromide, and visualized under UV light. Amplifications for SSR and ISSR were repeated twice. Genetic similarities between micropropagated and mother plants were measured by the Jaccard’s similarity coefficient (Jaccard, 1908) with the Past program. The similarity coefficients thus generated were used for constructing a dendrogram with a UPGMA option using the same Past program. 3. Results 3.1. In vitro multiplication The number of neoshoots from somaclones belonging to the same individual had a wide variability, with the multiplication rate ranging between 10 and 135 neoshoots/ inoculum, 90 days from inoculation. The average of shoot number, 110 days from inoculation (Figure 1) on 8 25 20 15 10 5 0

M1

M2 M3 M4 No. of shoots No. of roots

M5

M6 M7 M8 M9 Average length of shoots Average length of roots

Figure 1. The influence of culture medium on D. giganteus subsp. banaticus multiplication at 110 days from inoculation. M1 = K 0.1 mg/L, M2 = K 1.0 mg/L, M3 = BAP 0.1 mg/L, M4 = BAP 1.0 mg/L, M5 = 2iP 10 mg/L, M6 = 2iP 15 mg/L, M7 = TDZ 0.01 mg/L, M8 = TDZ 0.05 mg/L, and M9 = MS. Values represent means ± SD.

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culture media with hormones (and 1 without hormones), shows that with kinetin (K; M1 and M2 media) and thidiazurone (TDZ; M7 and M8) a higher concentration of the hormone (1.0 mg/L K and 0.05 mg/L TDZ) ensures a better multiplication rate (M1-12.38, M2-15, M7-12.27, M8-14.63; shoots/inoculum) than BAP (M3 and M4) and 2iP (M5 and M6), which ensure better multiplication at lower concentrations (M3-17.07, M4-16.5, M5-19, M612.5; shoots/inoculum), which we obtained on media supplemented with 0.1 mg/L BAP and 15 mg/L N6-[2isopentyl]adenine (2iP). The best multiplication 110 days from inoculation was obtained on M1, M2, M3, and M9 culture media (Figure 1). The root induction was also good on these media. According to statistical analysis of data 110 days after inoculation, there were no statistically significant differences between the 9 culture media regarding number of shoots. In contrast, regarding the length of shoots, statistically significant differences were found among different variants: M1-M2**, M1-M3*, M1-M4***, M1-M5***, M1-M6***, M1-M7***, M1-8***, M2-M5*, M2-M6***, M2-M7***, M2-M8***, M3-M5**, M3-M6***, M3-M7***, M3-M8***, M4-M8*, M4-M9***, M5-M9***, M6-M9***, M7-M9***, and M8-M9*** (*= P < 0.05, **= P < 0.01, ***= P < 0.001). Root induction was similar on all culture media with similar results obtained, and there were no statistically significant differences among the 4 culture media in terms of root length or number (R1-21.25, R2-23.27, R3-18.53, R4-22.2; roots/inoculums) (Figure 2). 3.2. ISSR analysis A total of 6 primers were tested, but successful amplification was obtained only with 4 primers. With primers BC 812 and BC 861 amplification failed although the alignment temperature was changed, probably because these primers did not exert complementarity on the Dianthus genome. 25 20 15 10 5 0

M1

M2 M3 M4 No. of shoots No. of roots

M5

M6 M7 M8 M9 Average length of shoots Average length of roots

Figure 2. The influence of culture medium on D. giganteus subsp. banaticus rhizogenesis 140 days from inoculation. R1 = MS, R2 = NAA 0.1 mg/L, R3 = NAA 1 mg/L, and R4 = IAA 1 mg/L. Values represent means ± SD.

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The possible explanation is that ISSR primers used in this study are not specific for Dianthus; they were previously used for peach. DNA analysis with ISSR markers (BC 809, BC 826, BC 873, and BC 878) shows high genetic polymorphism of Dianthus giganteus subsp. banaticus individuals (Figure 3). A total of 24 scorable and reproducible fragments were obtained, and among them 1 band was monomorphic (4.17%) and 23 bands were polymorphic (95.83%). Thus, amplification with primer BC 809 shows 7 different fragments, from which 200 bp is present in all individuals, both before and after conservation (Figure 3a). Thus, in this case there is a difference between individual 3.1 in the natural habitat and 3.1 after in vitro culture, as different fragments were obtained. Amplification with primer BC 26 gave a total of 6 different fragments (Figure 3b). Several differences between individuals from natural habitats and those preserved by in vitro culture were observed. In the case of individuals 3.1v and 3.3v amplification was not achieved. After amplification with primer BC 873 a total of 8 fragments of about 50 and 400 bp were obtained. These fragments appeared randomly in studied individuals and were missing in individuals 1.1v, 2.3v, 2.3v, and 3.1v (Figure 3c). For this primer several amplifications were performed, the annealing temperature was reduced, and the Mg concentration was increased, but amplification was not obtained. Amplification with BC 878 primer generated 3 fragments (80, 100, and 200 bp) that were identified in only 1–2 individuals in each population, and they were more common in preserved individuals than those from natural habitats (Figure 3d). 3.3. SSR analysis All 5 SSR primers produced clear and reproducible bands. A total of 7 scorable and reproducible fragments were obtained in plants from natural habitats; among these, 3 bands were monomorphic (42.86%), and 4 bands were polymorphic (57.14%). The SSR pattern of in vitro plants reveals differences. Thus, in these plants a total of 8 scorable, clear, and reproducible fragments were obtained; among these, 2 bands were monomorphic (25%), and 6 bands were polymorphic (75%). By amplification of DNA from natural habitat plants with MS-DCAMCRBSY primer, 1 allele of 125 bp was obtained. This allele is present in all individuals, independent of the population. By amplification with MS-DCDIA30 primer, 1 more fragment of about 175 bp was obtained (Figure 4a). The genetic polymorphism revealed by the MS-DCDIA30 primer is very low, and all the individuals show the same fragments, except individual 3.2, which does not show this fragment. Conservation by in vitro culture did not significantly influence the pattern of the fragments

JARDA et al. / Turk J Biol

a

b

c

d

Figure 3. Banding pattern obtained with ISSR primers in D. giganteus subsp. banaticus individuals from natural habitat and in vitro conserved. a) BC 809, b) BC 826, c) BC 873, d) BC 878 primers (m: molecular marker; 1.1, 1.2, 1.3, 2.1 2.2, 2.3 3.1, 3.2, 3.3, 1.1v, 1.2v, 1.3v, 2.2v, 2.3v, 3.1v, 3.3v, 1.2v, 2.3v, 3.1v); 1.1–1.3: individuals from Domogled population; 2.1–2.3: individuals from Porţile de Fier population; 3.1–3.3: individuals from Cheile Ţesnei Gorge population; v: in vitro plants. Separation on 1.0% agarose gel, stained with 0.5 μg/mL ethidium bromide.

obtained with these 2 primers. By MS-DCAMCRBSY primer 2 fragments were obtained (Figure 4b), one of them of about 100 bp and the second about 125 bp, which was present in plants from natural habitats. By amplification with MS-DCDIA30 primer the same allele of about 175 bp was obtained, but some of the individuals did not present this fragment (3.1v).

DNA amplification of in situ plants with MSDINCARACC primer showed 1 fragment of 200 bp that is present in all individuals independent of the population (Figure 5a). The SSR pattern of in vitro plants obtained with this primer is the same; thus, the fragment of 200 bp was obtained in most individuals, but it is missing in 3.1v (Figure 5b).

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a

b Figure 4. Banding pattern obtained with MS-DCAMCRBSY and MS-DCDIA30 primers in D. giganteus subsp. banaticus individuals from natural habitat (a) and in vitro conserved (b) (m: molecular marker; 1.1, 1.2, 1.3, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3, 1.1v, 1.2v, 1.3v, 2.2v, 2.3v, 3.1v, 3.3v, 1.2v, 2.3v, 3.1v); 1.1–1.3: individuals from Domogled population; 2.1–2.3: individuals from Porţile de Fier population; 3.1–3.3: individuals from Cheile Ţesnei Gorge population; v: in vitro plants. Separation on 1.5% agarose gel, stained with 0.5 μg/mL ethidium bromide.

a

b Figure 5. Banding pattern obtained with MS-DINCARACC and MS-DINGSTA primers in D. giganteus subsp. banaticus individuals from natural habitat (a) and in vitro conserved (b) (m: molecular marker; 1.1, 1.2, 1.3, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3, 1.1v, 1.2v, 1.3v, 2.2v, 2.3v, 3.1v, 3.3v, 1.2v, 2.3v, 3.1v); 1.1–1.3: individuals from Domogled population; 2.1–2.3: individuals from Porţile de Fier population; 3.1–3.3: individuals from Cheile Ţesnei Gorge population; v: in vitro plants. Separation on 1.5% agarose gel, stained with 0.5 μg/mL ethidium bromide.

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Amplification with MS-DINGSTA primer showed a very polymorphic pattern. Thus, 3 fragments were identified, one of them of about 250 bp is present in 2 individuals from population 1 (1.1, 1.3), the second fragment of about 200 bp is present only in the individuals belonging to population 2 (2.1, 2.3), and the third fragment of about 150 bp is present in 2 individuals from population 1 (1.1, 1.2) (Figure 5a). The genetic polymorphism observed with this primer also appeared in the conserved plantlets. In vitro plants showed the same 3 fragments (Figure 5b). The primer MS-DINMADSBOX showed 1 fragment of 150 bp that was present in all individuals from natural habitats as well as in vitro plants (Figure 6). 4. Discussion 4.1. In vitro multiplication The results regarding in vitro multiplication of this plant species are comparable with the previous results obtained by Cristea et al. (2006). They obtained 8 shoots/inoculum after 56 day of culture on medium with K and NAA (1.0 mg/L of each). Previous observations at 30 and 60 days from the transfer on D2 medium suggest that after 30 days the plants from all 3 populations multiplied relatively uniformly, but after 60 days there is an obvious difference between the populations (Jarda et al., 2011). A high multiplication rate (110–240 neoshoots/inoculum) after 83 days of culture on medium supplemented with BAP (1.0 mg/L) and NAA (0.1 mg/L) was also obtained for another strictly endemic species in the South Carpathians, D. henteri Heuff. ex Griseb. & Schenk (Cristea et al., 2010). Similar results were obtained by Pop and Pamfil (2011) in this species and another 2 Dianthus species. It is well known that sometimes culture media without phytohormones stimulate rhizogenesis, and many times

allow satisfactory in vitro multiplication (Kovac, 1995). Our results obtained on media without phytohormones were mostly similar with those obtained on media with hormones (Figures 1 and 2). Our results regarding root induction are comparable with those obtained for the same taxon by Cristea et al. (2006). They obtained 11 roots/inoculum after 56 days of culture on medium supplemented with K and NAA (1.0 mg/L each). The medium without hormones (M9) causes the induction of a lower number of shoots, but the inoculants have numerous roots (10.9 roots/inoculum), and this disagrees with the results of Pop and Pamfil (2011). They did not obtain roots on the same culture medium. There are few data regarding in vitro multiplication of rare or endangered species of Dianthus in Europe, and reports include D. giganteus subsp. croaticus (Prolic et al., 2002), D. gratianopolitanus (Fraga et al., 2004), D. pyrenaicus (Marcu et al., 2006), D. giganteus, D. alpinus, D. ferrugineus, and D. gallicus (Cristea et al., 2006). Previous studies focused on in vitro multiplication of Romanian endemic and/or endangered Dianthus species, including D. petraeus subsp. simonkaianus (Miclăuş et al., 2003), D. glacialis subsp. gelidus (Cristea et al., 2006), D. nardiformis (Holobiuc et al., 2009), D. henteri (Cristea et al., 2010), and D. giganteus subsp. banaticus (Cristea et al., 2006; Jarda et al., 2011). Our results show that Dianthus giganteus subsp. banaticus supports long in vitro culture passages (110–140 days) and can be preserved as a living collection. 4.2. Molecular analysis Molecular markers are valuable tools for identifying certain fragments from DNA sequences, and many studies were performed regarding comparison of these fragments (Gostimsky et al., 2005). Studies regarding assessment of somaclonal variability in D. giganteus subsp.

Figure 6. Banding pattern obtained with MS-DINMADSBOX primer in D. giganteus subsp. banaticus individuals from natural habitat and in vitro conserved (m: molecular marker; 1.1, 1.2, 1.3, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3, 1.1v, 1.2v, 1.3v, 2.2v, 2.3v, 3.1v, 3.3v, 1.2v, 2.3v, 3.1v); 1.1–1.3: individuals from Domogled population; 2.1–2.3: individuals from Porţile de Fier population; 3.1–3.3: individuals from Cheile Ţesnei Gorge population; v: in vitro plants. Separation on 1.5% agarose gel, stained with 0.5 μg/mL ethidium bromide.

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1

3.1µ 3.1µ 2.2 2.3 2.2µ 3.3 3.3µ 1.3 1.3µ 2.3 2.3µ 2.1 3.1 1.1µ 1.2µ 1.2µ 1.2 1.1 3.2

banaticus show some changes in the absence/presence of certain fragments in some individuals. There are several differences regarding SSR or ISSR patterns of plants from natural habitats and in-vitro–derived plantlets. In some instances we had more in vitro plantlets belonging to the same individual from natural habitats. Patterns of different in vitro plantlets derived from the same mother plant were not the same. Differences among the patterns of 1.2, 2.3, and 3.1 plants from natural habitats and the in vitro plantlets 1.2v, 2.3v, and 3.1v were also observed. Somaclonal variation could appear after a period of in vitro conservation, as shown in Figures 3–6. ISSR and SSR markers were used for calculation of genetic similarities between plants from natural habitats and plants preserved by in vitro culture. Jaccard’s coefficient between plants ranged from 0.68 to 0.72 (Figure 7), but similarities could also be observed among different individuals belonging to the same populations. This coefficient also showed differences among individuals from different populations. The individuals belonging to the 3 populations were divided into 2 distinct groups. As was previously shown, the major contribution to this difference was revealed by ISSR markers, which were more polymorphic than SSR markers. Similar studies done in Dictyospermum ovalifolium (Chandrika et al., 2008) also show variability in micropropagated plants, while in Nothapodytes foetida (Chandrika et al., 2010) the banding patterns obtained

0.9 0.8

Eml/

0.7 0.6 0.5 0.4 0.3 0.2 0.1

Figure 7. Dendrogram illustrating similarities among plants from natural habitats and preserved plantlets by UPGMA cluster analysis calculated by ISSR and SSR markers.

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with each primer were uniform with those of the original plant, and they were both acclimatized and reintroduced in their natural habitat. There are considerable data regarding genetic diversity of Dianthus accessions assessed by SRAP and ISSR markers (Fu et al., 2008) and genotyping of carnation varieties with SSR markers (Smulders et al., 2003), but unfortunately the genetic structure of populations of rare and endangered species of Dianthus from Romania was never analyzed previously. Since the topic for this study is part of a larger research project regarding outdoor culture of endemic/endangered species of the genus Dianthus in Romania, a rocky area for these species alone was set up in Botanical Garden Alexandru Borza, Babeş-Bolyai University, Cluj-Napoca. In vitro plants of D. giganteus subsp. banaticus previously maintained in growth chamber, greenhouse, and shelter were transferred to this rocky area. Comparing the influence of culture medium on in vitro multiplication and rhizogenesis of D. giganteus subsp. banaticus we observed that by using MS culture medium without phytohormones we obtained results comparable to those from culture media with phytohormones. Thus, when plant material for eventual repopulation purposes is needed, MS medium without phytohormones is recommended, thus reducing the cost. Study of somaclonal variability of this taxon shows changes in the number of fragments and the molecular weight between individuals from a natural habitat and in vitro plants. We also conclude that ISSR markers detected higher polymorphism than SSR markers. SSR markers generated with the primer MS-DINGSTA showed genetic polymorphism inside the population and between populations. SSR markers generated with the primer MSDCAMCRBSY showed genetic polymorphism between in situ plants and in vitro plants. Genetic differences between in situ plants and in vitro plants are low, as seen from the Jaccard’s coefficient values. Thus, in vitro plants could be used for outdoor collection, and, if necessary, for replanting in natural habitats. Acknowledgments This work was financed by the Sectorial Operational Programme-Development of Human Resources POSDRU 6/1.5/S/3—Doctoral Studies: Through Science Towards Society and with the support of a grant from the Romanian Ministry of Education and Research in the Parteneriate PN II Programme (CNMP), project 31-008/2007. The authors are thankful to Dr Irina Goia and Dr Magdalena Palada for their help in collecting plant material from the field.

JARDA et al. / Turk J Biol

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