Acta Botanica 2-2016 - za web.indd 244 ACTA BOT. CROAT. 75 (2), 2016 Acta Bot. Croat. 75 (2), 244–252, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0028 ISSN 0365-0588 eISSN 1847-8476 Micropropagation of the narrow endemic Hladnikia pastinacifolia (Apiaceae) Jana Ambrožič-Dolinšek1, 2, 3*, Terezija Ciringer1, Mitja Kaligarič1, 3 1 University of Maribor, Faculty of Natural Sciences and Mathematics, Koroška 160, Maribor, Slovenia 2 University of Maribor, Faculty of Education, Koroška 160 Maribor, Slovenia 3 University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoče, Slovenia Abstract – The monotypic Hladnikia pastinacifolia Rchb. is a narrow endemic species, with an extremely small distribution area in Slovenia, prone to any kind of threat that could lead to species extinction. Tissue culture techniques are proposed as a conservation measure for rapid propagation and ex-situ conservation. Tissue culture was initiated from seeds and juvenile plants obtained from natural sites on a solid Murashige and Skoog (MS) medium, with and without growth regulators. We tested various combinations and concen- trations of growth regulators, and the best proliferation of axillary shoots, on average 14, was obtained on MS medium with 5 μM BAP and 3 μM IBA and 3% sucrose. Rooting was achieved after transferral of the shoots to an MS medium with 2 μM IBA and 3% sucrose. The rooted plants were acclimatized on a mixture of lime- stone sand, potting soil and vermiculite in a ratio of 10:2:2, with pH in the range of 7.5–8.0. In vitro propaga- tion methods provide an important opportunity for the propagation and preservation of H. pastinacifolia by rapidly increasing the number of plants, without disturbing the wild population. Keywords: Apiaceae, biotechnology, conservation, endemic, Hladnikia pastinacifolia, micropropagation, tissue culture Abbreviation: BAP – 6-benzylaminopurine, IBA – indol-3-butyric acid, 2iP – 2-isopentinyl adenine, K – kine- tin, MS – Murashige and Skoog medium, NAA – naphthalene acetic acid, TDZ – thidiazuron, Z – zeatin * Corresponding author, e-mail: jana.ambrozic@um.si Introduction In the distant past and during more recent geological pe- riods, complex biogeographical and evolutionary processes resulted in an extraordinarily high number of endemic al- pine taxa (Berthouzoz et al. 2013). Five hundred and one endemic taxa have been described in the Alps, comprising ca. 9% of the 4,485 taxa inhabiting this mountain range (Aeschimann et al. 2011). The most emblematic species are the narrow endemics. However, only a few of them have been objects of intensive ecological and/or genetic studies (Berthouzoz et al. 2013). Hladnikia pastinacifolia Rchb. (Apiaceae) is one of the four endemic monotypic genera of the Alps – in addition to Berardia, Physoplexis and Rhizo- botrya (Wraber 2009). It has an extremely narrow distribu- tion area of 4 km2 within the high karst plateau of Trnovski gozd in Slovenia (Aeschimann et al. 2004), which repre- sents a bridge between the Alps and the North-Western Di- naric Alps. The species is also famous for its uniquely iso- lated phylogenetic position within the Apiaceae family (Šajna et al. 2012). It is not restricted to a specifi c niche; on the contrary, it thrives in a variety of the habitats (Šajna et al. 2012, 2014) that are available within the entire area of the High plateaus of the Dinaric Alps (Mayer 1960). It is still unknown why the distribution range is so narrow. Thus, the species’ inability to populate favorable habitats in an area wider than a few isolated populations within a few square kilometers is the reason for concern regarding its persistence and has placed the plant at risk. Rare narrow endemic species occurring in a few small populations have to cope with random genetic drift, inbreeding, a stronger founder effect, and a greater potential for demographic bot- tlenecks that result in low genetic variability (Gaston and Kunin 1997). Small habitat populations decrease the chanc- es of the plant outcrossing. The low potential for range ex- pansion and recolonization was additionally explained by poor seed dispersal (Šajna et al. 2012). Thus, along with all mentioned restrictions for its dispersal across appropriate habitats and potential for further adaptation, its unique tax- onomic position too makes H. pastinacifolia vulnerable and a potential candidate for extinction, especially on account MICROPROPAGATION OF ENDEMIC HLADNIKIA PASTINACIFOLIA ACTA BOT. CROAT. 75 (2), 2016 245 of the forecast global climatic changes (Dawson et al. 2011). Species that persist in small, isolated populations with low genetic diversity will have limited ability to adapt to new climate conditions (Lavergne et al. 2010). Hladnikia pastinacifolia has another peculiarity: the small distribution range is divided into two populations by a large dense forest, which is unsuitable as habitat, in be- tween. It has been presumed that such isolated distribution would have caused genetic divergence after the last glacia- tion. For that reason, its genetic variability has been tested, but divergence was not confi rmed. A general loss of genetic diversity has been proved with molecular techniques using RAPD markers (Šajna et al. 2012). The authors concluded that the genetically homogeneous populations of Hladnikia pastinacifolia are the result of severe bottlenecks, which dramatically reduced or eliminated some populations, re- gardless of the time of colonization (before or after glacia- tion); the extant populations were founded by a single lin- eage, starting from a founding population. The conservation status of Hladinika pastinacifolia is satisfactory. It is protected by the Decree on rare and threat- ened wild plant species (Offi cial Gazette of the Republic of Slovenia 2002, 2010), with a status 4, meaning that mea- sures for favorable conservation status on its habitat should be implemented. Furthermore, this species is listed as a “Natura 2000 species” in Annex II of the Habitat Directive (Council Directive 1992), which also envisages the desig- nation of a Natura 2000 site on the basis of its occurrence. There have already been some attempts at ex situ conserva- tion in the Botanical Garden of the University of Ljubljana, where a seed collection and living exemplars are stored. Both methods have disadvantages. One is connected with the short life cycle, since monocarpic H. pastincifolia (Šajna et al. 2009, 2011, 2014) fl owers once in its lifetime, and dies after fl owering. The other is connected with problem- atic seed germination established in the Apiaceae family (Hendrawati et al. 2012, Baskin and Baskin 2014). For such exceptional botanical rarities, it is nowadays usual to apply, besides the conventional propagation methods, in vitro propagation methods, which provide an important opportu- nity for the propagation and preservation of endemic, rare and/or endangered plant species in general (Fay 1992, Rao 2004, Pence 2010, 2011, Cruz-Cruz et al. 2013). Only in cases where a previously optimized and tested propagation protocol exists would this kind of plant escape extinction from sudden changes in natural conditions or other factors that place the plant at risk of local extinction (which is here the defi nitive level). Over-collecting and competition with other species, along with reduction and fragmentation or degradation of natural habitats could di- minish the population substantially. Micropropagation is a convenient measure suggested for preserving endangered species by rapidly increasing the number of plants in the case of natural population loss in a relatively short period of time and introducing them to their natural or to new envi- ronments (Sarasan et al. 2006, Kapai et al. 2010, Cruz-Cruz et al. 2013, González-Benito and Martín 2011). Beside this, tissue culture allows genotype conservation (Tavares et al. 2010b), constitutes a kind of living ex situ collection and is a basic method for other biotechnological conservation measures like cryopreservation of shoots (Cruz-Cruz et al. 2013). Several institutions, like the Kew and Missouri Bo- tanical Gardens (Sarasan et al. 2006), and the Botanic Gar- den of the University of Coimbra for endemic Apiaceae, are now using this strategy to propagate and maintain endan- gered and endemic species (Tavares et al. 2009–2010, 2010b). The problems with such propagation include the potential for somaclonal variation, as a consequence of ei- ther genetic or epigenetic changes in the tissue culture- propagated material (Bairu et al. 2011). To obtain plant ma- terial that is as genetically stable and uniform as possible, with a low probability of genetic or epigenetic variation, micropropagation must be carried out through shoot tip or axillary bud proliferation (Pierik 1991). The aim of the present work was to establish and evalu- ate the in vitro propagation protocol for Hladnikia pasti- nacifolia through axillary shoots in order to obtain enough plants that can be used for several purposes allowed be- cause of legislative restrictions connected with sample quantities and for establishment of ex situ collections in bo- tanical gardens, production of seeds for seed banking, rein- troduction to the natural environment, for basic research on the ecology and biology of threatened plant species, and for long-term maintenance in a tissue culture collection – all, without disturbing the wild population. Material and methods Plant material, explant source and culture condition All plant material, seeds and juvenile plants were col- lected at natural secondary sites near the route to Predmeja, Slovenia (Figs. 1a, b), with the permission of the responsi- ble authority in 2004 and 2013. Seeds and juvenile plants of Hladnikia pastinacifolia were used as initial explants (Figs. 1c, d). Since the species is protected by law, we performed only one culture initiation by seed germination, and one ini- tiation of the culture from the juvenile plants collected at the natural sites in 2004. Tissue culture was initiated once more in 2013, with sterilized juvenile plants collected at the natural sites. Each plant was the source of one tissue culture line for the propagation of plant material. Seeds and all plant material were surface sterilized with 70% ethanol (Sigma Aldrich) for 30 seconds and then soaked in 1% or 2% commercial bleach 6.7% NaOCl (Šam- pionka), with a drop of the detergent Tween 80 (Merck) for 15–20 min and rinsed three times with sterile deionized water . We tested several factors to increase the germination rate: cold treatment, scarifi cation and sterilization of seeds. Sterilized and non-sterilized seeds germinated in sterile pe- tri-dishes closed with parafi lm (Bemis), on moistened paper bridges at 20 °C in the growth chamber or in the fridge at 4 °C in the dark. The cultures in the growth chamber were kept at 23 ± 2 °C, with a photoperiod of 16 h at 38–50 μmol m–2 s–1 (Osram L 58W/77 – Fluora) and 50% relative hu- midity. AMBROŽIČ-DOLINŠEK J., CIRINGER T., KALIGARIČ M. 246 ACTA BOT. CROAT. 75 (2), 2016 Culture medium for shoot proliferation and multiplication In 2004 and the next two years, tissue culture was initi- ated from 12 sterilized juvenile plants from the natural sites and 10 sprouts from seeds (Figs. 1c, d), and the best con- centrations were additionally tested on juvenile plants from the natural site in 2013. The sterilized juvenile plants and sprouts were placed on solid MS medium (Murashige and Skoog 1962). supplemented with 0.8% Difco-bacto-agar (Medias International), with 3% sucrose (Duchefa Bioche- mie), with and without growth regulators: 6-benzylamino- purine (0–20 μM BAP, Sigma Aldrich), 2-isopentinyl ade- nine (0–20 μM 2iP, Sigma Aldrich), thidiazuron (0–10 μM TDZ, Sigma Aldrich), kinetin (0–20 μM K, Sigma Aldrih), zeatin (0–20 μM Z, Sigma Aldrich), naphthalene acetic acid (0–1 μM NAA, Sigma Aldrich), indol-3-butyric acid (0–3μM IBA, Sigma Aldrich) (Tab. 1) adjusted to pH 5.7– 5.8, and later on autoclaved. Root induction and acclimatization The regenerated shoots were rooted on MS media with different combinations and concentrations of the growth regulators BAP (0–1 μM) and IBA (2–10 μM). In the root- ing experiment, the following combinations and concentra- tions of the growth regulators were tested: 2 μM IBA and 1 μM BAP; 5 μM IBA and 1 μM BAP; 10 μM IBA and 1 μM BAP; 5μM IBA and 2 μM BAP; 10 μM IBA a nd 2 μM BAP; 2 μM IBA and 0 μM BAP; 5 μM IBA and 0 μM BAP; 10 μM IBA and 0 μM BAP. Rooted plants were transferred to the prepared substrate containing a mixture of limestone sand : potting soil : ver- miculite (10:1:2) with a pH of 7.0–7.5, or in a mixture of limestone sand : potting soil : vermiculite (Vermit Group) (10:2:2) with a pH of 7.5–8.5, and transferred to in vivo greenhouse conditions. The plants were protected against excessive water loss by regular spraying with water, and by plastic covers with adjustable aeration openings. They were acclimatized with a progressive increase in aeration through the adjustable aeration openings for the fi rst two weeks, and with progressive opening of the whole cover over the next three weeks. Statistical analysis The statistical package SPSS® 21.0 was used for data analysis. Student’s t-test and the 2 x 2 Chi-squared test (χ2) were used for evaluating levels of statistical signifi cance (P) between treatments. Statistical signifi cance was shown between control on MS without and with growth regulators, unless otherwise denoted. The symbols used in the fi gures are as follows: NS denotes not signifi cant, symbol (*) de- notes P < 0.05, symbol (**) denotes P < 0.01, symbol (***) denotes P < 0.001. All experiments were repeated twice with similar results. Fig. 1. Micropropagation of Hladnikia pastinacifolia: a, b) plants from the natural habitat in Predmeja (bar = 5 cm); c) sterilized seeds germinated into sprouts (bar = 1 cm); d) initiation of the culture with sprouts from natural sites (bar = 1 cm); e) the shoots developed on MS medium with 5 μM 6-benzylaminopurine (BAP) and 3 μM – indol-3-butyric acid (IBA); f) detached juvenile shoots are the most suit- able for multiplication of H. pastinacifolia shoots; g) the effect of BAP on the length of shoots (bar = 2.5 cm); h) fl ower shoots developed sporadically from vegetative shoots (bar = 2.5 cm); i) rooted H. pastinacifolia shoots after 4 weeks on MS medium with 2 μM BAP (bar = 2.5 cm); j) rooted plants transferred to plastic pots with substrate for acclimatization (bar = 2.5 cm); k) acclimated plants after 6 months at outdoor conditions (bar = 1 cm). MICROPROPAGATION OF ENDEMIC HLADNIKIA PASTINACIFOLIA ACTA BOT. CROAT. 75 (2), 2016 247 Tab. 1. Effect of different cytokinins and auxins on shoot development of detached shoots of Hladnikia pastinacifolia after two months of culture. BAP – 6-benzylaminopurine, 2iP – 2-isopentinyl adenine, TDZ – thidiazuron, K – kinetin, Z – zeatin, IBA – indol-3-butyric acid, 2,4-D – 2,4-dichlorophenoxyacetic acid, NAA – naphthalene acetic acid, SD – standard deviation. * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001. Signifi cance was shown between shoots on MS without and with hormones, except if it is denoted different: awith 2 μM BAP; bwith 5 μM BAP; cwith 10 μM BAP; dwith 0 μM TDZ. Hormones [μM] Explants with new shoots [%] Average number of shoots ± SDBAP 2iP TDZ KIN Z IBA 2,4-D NAA 0 0 0 0 0 0 0 0 9.3 3.7±0.4 2 0 0 0 0 0 0 0 27.3 6.4±1.5 * a 2 0 0 0 0 2 0 0 69.5 10.0±1.9 * a 2 0 0 0 0 3 0 0 71.0 9.1±2.5 * a 2 0 0 0 0 0 3 0 – callus 2 0 0 0 0 0 5 0 – callus 2 0 0 0 0 0 10 0 – callus 5 0 0 0 0 0 0 0.5 25.0 6.8±1.1 ** 5 0 0 0 0 0 0 1 10.3 6.1±0.9 * 5 0 0 0 0 0 0 0 38.5 6.6±0.7*** b 5 0 0 0 0 0.5 0 0 37.0 5.8±0.7 ** 5 0 0 0 0 1 0 0 39.7 5.2±0.2 ** 5 0 0 0 0 2 0 0 68.0 10.5±1. 6 ** b 5 0 0 0 0 3 0 0 90.2 14.0±2.7 ** b 5 0 0 0 0 0 10 0 – callus 10 0 0 0 0 0 0 0 23.3 7.7±1.1 ** c 10 0 0 0 0 0 0 0.5 57.0 6.5±1.0 * 10 0 0 0 0 0 0 1 43.5 6.3±0.4 *** 10 0 0 0 0 0.5 0 0 35.0 6.0±0.6 ** 10 0 0 0 0 1 0 0 55.3 7.3±1.4 *** 10 0 0 0 0 2 0 0 67.3 12.0±2.2 ** c 10 0 0 0 0 3 0 0 72.7 10.1±2.3 * c 20 0 0 0 0 0 0 0 17.0 5.6±0.3 *** 20 0 0 0 0 3 0 0 53.3 8.5±2.1 0 2 0 0 0 0 0 0 10.3 4.5±0.7 0 5 0 0 0 0 0 0 20.0 6.3±1.4 * 0 10 0 0 0 0 0 0 39.0 8.1±1.7 ** 0 10 0 0 0 0 0 0.5 40.0 6.8±1.6 * 0 10 0 0 0 0.5 0 0 27.3 5.3±1.0 0 20 0 0 0 0 0 0 50.3 8.6±2.0 ** 0 20 0 0 0 0 0 0.5 34.7 4.7±0.5 0 20 0 0 0 0.5 0 0 38.0 5.4±1.8 * 0 0 0.25 0 0 0 0 0 46.3 7.3±1.7 * 0 0 0.5 0 0 0 0 0 74.3 8.50±2.12 * 0 0 0.5 0 0 2 0 0 73.7 9.81±1.5 *** 0 0 1 0 0 0 0 0 34.7 5.1±1.1 0 0 2 0 0 0 0 0 39.0 6.2±1.0 * 0 0 5 0 0 0 0 0 26.3 5.2±0.8 0 0 10 0 0 0 0 0 27.0 4.3±0.7 0 0 0 1 0 0 0 0 6.3 3.6±0.3 0 0 0 2 0 0 0 0 14.0 4.1±0.6 0 0 0 5 0 0 0 0 14.0 4.0±0.3 0 0 0 10 0 0 0 0 11.3 3.8±0.4 0 0 0 20 0 0 0 0 13.3 4.2±0.7 0 0 0 0 0.5 0 0 0 8.5 3.6±0.3 0 0 0 0 2 0 0 0 21.0 5.5±1.2 0 0 0 0 3 0 0 0 21.0 5.2±1.4 0 0 0 0 5 0 0 0 28.0 4.5±0.6 0 0 0 0 10 0 0 0 52.5 4.6±2.2 * 0 0 0 0 20 0 0 0 34.0 4.1±0.1 0 0 0 0 0 0 1 0 callus 0 0 0 0 0 0 3 0 callus 0 0 0 0 0 0 5 0 callus 0 0 0 0 0 0 10 0 callus AMBROŽIČ-DOLINŠEK J., CIRINGER T., KALIGARIČ M. 248 ACTA BOT. CROAT. 75 (2), 2016 Results In vitro seed germination and initiation of the culture The seeds of H. pastinacifolia started to germinate after 4 months in the culture (Fig. 1c) and achieved their highest germination rate after one year. We tried several factors which could infl uence the germination rate: room tempera- ture and cold treatment, scarifi cation and sterilization of seeds. Only those seeds under permanent cold treatment germinated but failed to germinate at all if they were kept in the growth chamber. Scarifi cation did not promote germi- nation. Between 7% of the sterilized and 30% of the unster- ilized seeds germinated after 4 months and between 45% of the sterilized and 60% of the unsterilized seeds germinated after one year of cold treatment. Sterilization decreased and slowed the rate of early germination. This was signifi cantly evident (χ2 = 10.960; df = 1; P = 0.001) after four months of germination but no longer signifi cantly evident after one year of germination. Tissue culture was initiated from shoots excised from juvenile sprouts (Fig. 1c) germinated from sterilized seeds, from sterilized juvenile sprouts ger- minated from the unsterilized seeds and from juvenile plants from the natural sites (Fig. 1d). Shoot proliferation and multiplication Permission for the application, which allowed only a small number of explants, limited our research. That is why we began our experiments with a small number of initial explants. In these early experiments we fi rst cultivated a small number of explants on MS medium with and without 2–20 μM BAP (data not shown); this was later repeated several times on a higher numbers of explants (Fig. 2a). Shoot multiplication was observed on all MS media, with or without growth regulators. Shoot proliferation was opti- mal when 5 and 10 μM BAP were used, and the best growth medium produced on average, 7.3 shoots per explant (Fig. 2a). This plant material was used for further experiments, where several concentrations and combinations of cytoki- nins and auxins were tested (Tab. 1). In further experiments, the development of new shoots was observed on almost all media tested (Tab. 1). There was a signifi cant difference within the various treatments using different combinations and concentrations of growth regulators. The addition of auxins to cytokinins additionally improved shoot development (Tab. 1). New shoots devel- oped on almost all the explants. The best proliferation of shoots, on average 14 (Fig. 1e, f, g), was obtained on MS medium with 5 μM BAP and 3 μM IBA (Fig. 2b, Tab. 1). Shoots developed on 90% of explants. Slightly fewer shoots, on average 12, were obtained on MS medium with 10 μM BAP and 2 μM IBA (Tab. 1). Shoots developed on 67% of these explants. The new shoots began to proliferate after 10 to 14 days. Over the next 15 to 20 days, the shoots elongat- ed to a length appropriate for detachment and rooting. These detached shoots exhibited both organized and un- organized growth (Tab. 1). Unorganized growth was ob- served only on the MS medium with growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) (Tab. 1). Flowers developed sporadically from the new shoots. Well-devel- oped fl ower shoots formed several infl orescences at the shoot tip, which developed into a considerable number of fl owers (Fig. 1h). Growth regulators did infl uence the shoot length. With an increased concentration of BAP, smaller shoots devel- oped, if BAP alone was added to the media. This was sig- nifi cantly evident when concentrations exceeded 5 μM BAP (Fig. 1g). Root development and acclimatization Roots did not develop on those media without growth regulators. To obtain roots, we tested several combinations and concentrations of growth regulators. We started by de- creasing the amounts of cytokinin BAP and increasing the auxin IBA and fi nished by increasing only the auxin IBA. Fig. 2. Effect of plant growth regulators on the shoot and root de- velopment in the micropropagation of Hladnikia pastinacifolia: a) the effect of 6-benzylaminopurine (BAP) on the number of shoots (n = 28) after 35 days of culture; b) the effect of BAP and indol-3- butyric acid (IBA) on the number of shoots (n = 28) after 35 days of culture; c) root development (n = 25) on MS media supple- mented by different concentrations of IBA after 4 weeks of cul- ture. Statistically signifi cant differences (t-test) are shown between the control without hormones (a, b) or MS medium with 2 μM IBA (c) and different treatments. SD -standard deviation, * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001. MICROPROPAGATION OF ENDEMIC HLADNIKIA PASTINACIFOLIA ACTA BOT. CROAT. 75 (2), 2016 249 The best rooting was achieved on the MS medium with IBA alone (Fig.. 1i, 2c). Roots started to develop after 2 weeks. Optimal rooting was obtained on the MS medium with 2 μM or 5 μM IBA after 4 weeks of culture. The great- est number, 44% of shoots with roots, was obtained on the MS medium with 2 μM BAP and the smallest, 28% of shoots with roots, was obtained on the MS medium with 5μM IBA. Shoots with roots developed an average 10.8 of roots on medium with 2 μM IBA. A lower average of 6.9 roots developed on the medium with 5 μM IBA, and the signifi cantly lower average of 2.6 roots appeared on the MS medium with 10 μM IBA. The higher concentration, 10 μM IBA, induced both roots and callus to develop. Roots devel- oped on the detached side of the shoots. Two growth substrates with different pH were assessed for effi ciency in supporting ex vitro growth. Both mixtures are attempts to imitate conditions in the natural habitat. They were prepared with limestone sand, which increases the pH of the substrate, potting soil and vermiculite. After 5 months, 66% of the plants survived in the substrate with a higher pH between 7.5 and 8.5 (Fig. 1j) and only 22% in substrate with a lower pH between 7 and 7.5. Plants of H. pastiancifolia obtained by this procedure are transferred to outside conditions (Fig. 1k). Discussion Like many other alpine plant endemics (Aeschimann et al. 2011), Hladnikia pastinacifolia, has an extremely re- stricted distribution. Due to its inability to colonize appro- priate habitats in a wider range due to several reasons, due to genetic depauperation, which make its capacity of adap- tation to climate change even weaker, Hladnikia pastinaci- folia is a strictly protected species (Šajna et al. 2012). This is the reason for the restrictions on the collecting of seeds for seed banks and research activities. Micropropagation of H. pastinacifolia provides the possibility for conservation in tissue culture collection, without disturbing the wild pop- ulation and allows rapid propagation of H. pastinacifolia for research efforts. Seed-banking is the primary method for ex-situ conser- vation. We had shown that H. pastinacifolia had problem- atic seed germination. Seeds from cold wet environments have been shown to be relatively short lived in storage, and successful long-term seed conservation for alpine plants may be diffi cult (Mondoni et al. 2011). Therefore H. pasti- nacifolia has to be conserved either in living collections or through micropropagation and/or cryogenic storage, and this article presents the latter method. In vitro seed germination and initiation of the culture Micropropagation was started with the introduction of plant material in aseptic conditions. We started with limited sample quantities and with different sources of plant mate- rial. Seeds, juvenile sprouts from seeds, and juvenile plants of H. pastinacifolia were used as initial explants for initia- tion of the culture. The starting material had a range of dis- advantages connected with germination and/or the estab- lishment of aseptic culture conditions. H. pastinacifolia has problematic seed germination. These seeds did not germinate in conditions that usually promote germination. When they did germinate, they did so neither in high percentages nor immediately and simultane- ously. For that reason, the initiation of culture from seeds was time consuming, uncertain in outcome and in need of further consideration. The seeds began to germinate be- tween four and twelve months after initiation of the culture. The seeds in our experimental system germinated only after cold treatment and did not germinate at all in growth cham- ber conditions. The reason for this late and low germination could be dormancy, which is not unusual for seeds of the Apiaceae family (Hendrawati et al. 2012, Baskin and Baskin 2014), or possible loss of viability (Makunga et al. 2003, Mondoni et al. 2011). Sterilization only slowed the rate of early germination and slightly infl uences the fi nal germination of cold treated seeds. That is why the initiation of the culture through seeds or juvenile sprouts from seeds could be problematic, since this lengthens the initial stage of micropropagation by several months. The introduction of unsterilized plant material in aseptic conditions was also problematic, because of damage during sterilization. When we started the culture with sterilized seeds, they germinated into viable, juvenile sprouts. When we started the culture with unsterilized seeds that germinated into unsterilized ju- venile sprouts, there were losses due to contamination and/ or mechanical damage during their transfer to aseptic con- ditions by sterilization. Problems with the establishment of an aseptic culture make seeds more appropriate than juve- nile sprouts from seeds. When tissue culture was initiated from sterilized juve- nile plants from the natural sites, the initiation of the culture was problematic because sterilization did not always re- move contamination. These sources of contamination in- clude surface sterilization resistant plant endophytic micro- organisms and common environmental microorganisms, both of which may become pathogenic in culture (Cassells 2012). Contamination can resist sterilization and sooner or later damage plant material. Cover contamination makes juvenile plants taken from natural sites the least appropriate for tissue culture initiation. Shoot multiplication The development of new shoots was observed on al- most all media tested, even on the MS medium free of growth regulators. Growth regulators on optimal media more than tripled the number of shoots. The best prolifera- tion of shoots was obtained on the MS medium with 5 μM BAP and 3 μM, and slightly worse on the MS medium with 10 μM BAP and 2 μM IBA. A combination of BAP and IBA was used for shoot proliferation of Apiaceae Anethum graveolens (Sharma et al. 2004) and Centella asiatica (Ba- nerjee et al. 1999), fi rst in a combination of 2.2 μM BAP and 0.5 μM IBA, and second in a combination of 8.9 μM BAP and 0.5 μM IBA (Banerjee et al. 1999, Sharma et al. 2004). Other researchers (Hosain et al. 2000, Nath and Buragohain 2003, Sharma and Wakhlu 2003, Karuppusamy et al. 2007, Das et al. 2008, Jaheduzzaman et al. 2012) used a combination of BAP and NAA or a combination of BAP AMBROŽIČ-DOLINŠEK J., CIRINGER T., KALIGARIČ M. 250 ACTA BOT. CROAT. 75 (2), 2016 and IAA (Gaddaguti et al. 2013) for proliferation of shoots in Apiaceaes and an optimal concentration ranging from 2 to 17.5 μM of BAP and from 0.5 to 2.7 μM of NAA. The combination of BAP with NAA in our case was not as ef- fective in shoot regeneration. Growth regulators infl uence the shoot length of H. pas- tinacifolia. With an increased concentration of BAP, when BAP alone was added to the media, smaller shoots devel- oped. However, the trend toward shoot lowering was not evident when BAP was combined with different concentra- tions of auxin IBA (data not shown). The opposite trend was observed when 2iP alone was added to the media, where with the increased concentration of 2iP, taller shoots developed (data not shown). Sporadically observed fl ower development, instead of vegetative shoots, indicates the end of the life cycle for these plants. Since it occurred infrequently, it did not dis- turb the micropropagation procedure. Flower shoots with infl orescences were previously observed in Apiaceae tissue culture (Tavares et al. 2010a). Root initiation Rooting of shoots during propagation is genotype-de- pendent, and shoots of some species which root naturally without a rooting stage need a special rooting medium, with or without growth regulators. Hladnikia pastinatifolia shoots developed roots only on the MS medium with growth regulators. Optimal rooting was obtained on MS medium with 2 μM IBA. Concentrations higher than 5 μM IBA induced both roots and the development of callus. Root development with an intermediary callus indicated that at that roots and shoots were not well connected with vascular tissue and that plants are insuffi ciently supplied with nutri- ents (George 1993). Successful rooting in other members of the Apiaceae family was also achieved with 1–5 μM IBA (Tavares et al. 2010a, Das et al. 2008, Jana and Shekhawat 2011, Coste et al. 2012, Jaheduzzaman et al. 2012), and with higher concentrations of IBA (Hossain et al. 2000, Sharma and Wakhlu 2001, Banerjee et al. 1999, Tavares et al. 2009–2010), with NAA or IAA (Karuppusamy et al. 2007, Jaheduzzaman et al. 2012) and only rarely without growth regulators (Makunga et al. 2003). Acclimatization and transfer to in vivo conditions Acclimatization of H. pastinacifolia required a specially prepared substrate. The substrates usually used for acclima- tization with other members of that family (Sharma and Wakhlu 2001, Nath and Buragohain 2003, Tavares et al. 2010a, Coste et al. 2012, Hendrawati et al. 2012, Jaheduz- zaman et al. 2012) were far different from the substrate which promoted acclimatization of our plant species. H. pastinacifolia did not acclimatize at all if the substrates were rich in organic matter (data not shown). That is why we tested two artifi cial substrates, which imitated the crum- bling granulated texture and the alkaline nature of the sub- strate from the natural sites (Čušin 2004). We used two mixtures composed of limestone sand, potting soil and ver- miculite in different ratios. Our plants survived better in the substrate with a more alkaline reaction, with a pH between 7.5 and 8.5. Nevertheless, less than half the shoots were successfully acclimatized. Plants of H. pastiancifolia ob- tained by this procedure are now growing in outside condi- tions, showing the potential of this method to increase the number of plants available for conservation purposes. Do we need alternative ex situ conservation programs for alpine plants, especially endemic species like H. pasti- nacifolia? Alpine plants and their habitats are extremely fragile, easily disturbed and prone to any kind of climate change effects. During the past few decades, human activi- ty has increased in the alpine environment, and disturbance is probably the greatest threat to alpine plants. Protected ar- eas with specifi c conservation measures are an effi cient way to protect Alpine biodiversity, but uncertainty still per- sists, owing to a range of different climate-change driven effects. More than half the species growing within the Eu- ropean Natura 2000 network might lose suitable habitats in these areas. Hence, it will be necessary to prepare alterna- tive ex situ measures (Vittoz et al. 2013), of which the mi- cropropagation presented in this paper is claimed to be the most effi cient. Conclusion As an ancient paleoendemic and one of the most re- markable among the Pleistocene survivors, H. pastinacifo- lia is of great importance for understanding the effects of changing environment and deserves further research efforts in the direction of biodiversity studies and conservation programs (Šajna 2012). In our study we established a proto- col for in vitro propagation of Hladnikia pastinacifolia, from preparation of plant material, establishment of aseptic culture, multiplication, in vitro rooting, transplanting and acclimatization of plants. Furthermore, tissue culture col- lection has been successfully propagated continuously since 2006. We have maintained it for 10 years without loss of either juvenile character or shoot and root proliferation ca- pacity, the only exception being the sporadically observed fl ower development, which indicates the end of the life cy- cle for these plants. Since the rest of the material retained its juvenility, we can confi rm that we did achieve the goal of having a long-term collection of H. pastinacifolia in tis- sue culture. In 2013, we additionally started a tissue culture of H. pastinacifolia and obtained several new lines. With these lines, we additionally tested the pre-designed micro- propagation protocol, with repeated results. The tissue culture of H. pastinacifolia represents a liv- ing collection or back-up gene pool of this species. New plants can be used for several purposes: reintroduction to nature, assisted migration, strengthening the natural popu- lation, for ex situ collections in botanical or rock gardens, for studies on the ecology and biology of species, for its nutritional value, or simply for educational and ornamental purposes. Further studies on this species will focus on other specifi c biotechnological tools successfully used in several programs of plant conservation, like assessment of the de- gree of genetic variability of plants in culture and introduc- tion of techniques for long-term storage at low tempera- tures. MICROPROPAGATION OF ENDEMIC HLADNIKIA PASTINACIFOLIA ACTA BOT. CROAT. 75 (2), 2016 251 Acknowledgements We would like to thank Nina Šajna for collecting plant material at the natural sites. This research was supported by the Slovene Ministry of Higher Education, Science and Technology within the program Research to ensure food safety and health within the Grant No. P1-0164, lead by D. Škorjanc. References Aeschimann, D., Lauber, K., Moser, D. M., Theurillat, J. P., 2004: Flora alpina: ein atlas sämtlicher 4500 gefäßpfl anzen der Al- pen, 1108–1109. Haupt, cop. Bern, Stuttgart, Wien. Aeschimann, D., Rasolofo, N., Theurillat, J. P., 2011: Analyse de la fl ore des Alpes. 1: historique et biodiversité. Candollea 66, 27–55. Bairu, M. W., Aremu, A. O., Van Staden, J., 2011: Somaclonal variation in plants: causes and detection methods. Plant Growth Regulation 63, 147–173. Banerjee, S., Zehra, M., Kuma, S., 1999: In vitro multiplication of Centrella asiatica, a medical herb from leaf explants. Current Science 76, 147–148. Baskin, C. C., Baskin, J. M., 2014: Seeds: ecology, biogeography, and evolution of dormancy and germination 2nd edn. Aca- demic Press, Elsevier, Oxford. Berthouzoz, M., Maendly, S., Bétrisey, S., Mangili, S., Prunier, P., Lexer, C., Kozlowski, G., 2013: Some like it cold: distribu- tion, ecology and phylogeny of Arenaria bernensis Favarger (Caryophyllaceae) from the western Prealps in Switzerland. Alpine Botany 123, 65–75. Cassells, A. C., 2012: Pathogen and biological contamination management in plant tissue culture: phytopathogens, vitro pathogens, and vitro pests. In: Loyola-Vargas, V. M., Ochoa- Alejo, N. (ed.) Plant cell culture protocols, 57–80. Springer Science+Business Media. New York. Coste, A., Halmagy, A., Buticu-Keul, A. L., Deliu, C., Coldea, G., Hurdu, B. 2012: In vitro propagation and cryopreservation of Romanian endemic and rare Hypericum species. Plant Cell Tissue and Organ Culture 110, 213–226. Council Directive, 1992: Council Directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and fl ora. Retrieved August 14, 2014 from http://ec.europa.eu/ environment/nature/legislation/habitatsdirective/index_en. htm Cruz-Cruz, C. A., González-Arnao, M. T., Engelmann, F., 2013: Biotechnology and conservation of plant biodiversity. Re- sources 2, 73–95. Čušin, B., 2004: Hladnikia pastinacifolia Rchb. In: Čušin, B., (ed.) Natura 2000 in Slovenia – Plants, 107–113. ZRC SAZU, Ljubljana (In Slovenian). Das, R., Hasan, M. F., Hossain, M. S., Rahman, M., 2008: Micro- propagation of Centrella asiatica L. an important medicinal herb. Progressive Agriculture 19, 51–56. Dawson, T. P., Jackson, S. T., House, J. I., Prentice, I. C., Mace, G. M., 2011: Beyond predictions: biodiversity conservation in a changing climate. Science 332, 53–58. Fay, M. F., 1992: Conservation of rare and endangered plants us- ing in vitro methods. In Vitro Cellular & Developmental Biol- ogy – Plant 28, 1–4. Gaddaguti, V., Reddy, K. S., Deepthi, R. S., Lavanya, K., Rama, C., Talluri, V. R., Allu, P. R., 2013: In vitro fl ower induction and multiple shoot regeneration studies in Centella asiatica from nodal and leaf explants. Annals of Plant Sciences 2, 55– 58. Gaston, K. J., Kunin, W. E., 1997: What is rarity? In: Kunin, W. E., Gaston, K. J., (ed.) The biology of rarity, causes and conse- quences of rare-common differences, 30–47. Chapman and Hall, London. George, E. F., 1993: Plant propagation by tissue culture; Part 2: in practice, 2nd edn. Exegetics, Edington. González-Benito, M, E., Martín, C., 2011: In vitro preservation of spanish biodiversity. In Vitro Cellular and Developmental Bi- ology – Plant 47, 46–54. Hendrawati, O., Hille, J., Woerdenbag, H. J., Quax, W J, Kayser, O., 2012: In vitro regeneration of wild chervil (Anthriscus syl- vestris L.). In Vitro Cellular and Developmental Biology – Plant 48, 355–361. Hossain, S. N., Rahman, S., Joydhar, A., Islam, S., Hossain, M., 2000: In vitro propagation of thankuni (Centella asiatica L.). Plant Tissue Culture and Biotechnology 10, 17–23. Jaheduzzaman, M. D., Habib, M. A., Akter, S., Banu, N. A., Rah- man, R. B., Banu, T. A., Khan, S., Islam, S., 2012: In vitro re- generation of an important medicinal plant Centella asiatica L. Urban. Bangladesh Journal of Scientifi c and Industrial Re- search 47, 269–272. Jana, S., Shekhawat, G. S., 2011: Plant growth regulators, adenine sulfate and carbohydrates regulate organogenesis and in vitro fl owering of Anethum graveolens. Acta Physiologiae Planta- rum 33, 305–311. Kapai, V. Y., Kapoor, P., Rao, I. U., 2010: In vitro propagation for conservation of rare and threatened plants of India–a review. International Journal of Biological Technology 1, 1–14. Karuppusamy, S., Aruna, V., Kiranmai, C., Pullaiah, T., 2007: In vitro propagation of an endemic umbellifer, Hydrocotyle con- ferta. Indian Journal of Biotechnology 6, 541–544. Lavergne, S., Mouquet, N., Thuiller, W., Ronce, O., 2010: Biodi- versity and climate change: Integrating evolutionary and eco- logical responses of species and communities. Annual Review of Ecology, Evolution, and Systematics 41, 321–350. Makunga, N. P., Jäger, K. G., Van Staden, J., 2003: Micropropaga- tion of Thapsia garganica-a medicinal plant. Plant Cell Re- ports 21, 967–973. Mayer, E., 1960: Endemic fl owering plants in the southeastern limestone Alps, their foothills and transitional Illyrian territo- ry. In: Lazar, J. (ed.) Proceedings on the 150th anniversary of the Ljubljana Botanical Garden. Univerza v Ljubljani, Lju- bljana (in Slovenian). Mondoni, A., Probert, R. J., Rossi, G., Vegini, E., Hay, F. R., 2011: Seeds of alpine plants are short lived: implications for long- term conservation. Annals of Botany 107, 171–179. Murashige, T., Skoog, F., 1962: A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiolo- gia Plantarum 15, 473–497. Nath, S., Buragohain, A. K., 2003. In vitro method for propaga- tion of Centrella asiatica (L) Urban by shoot tip culture. Jour- nal of Plant Biochemistry and Biotechnology 12, 167–169. Offi cial Gazette of the Republic of Slovenia, 2002: Offi cial Ga- zette of the Republic of Slovenia, 82/02. Retrieved September 24, 2013 from http://www.uradni-list.si/1/objava.jsp?urlurid= 20024055 AMBROŽIČ-DOLINŠEK J., CIRINGER T., KALIGARIČ M. 252 ACTA BOT. CROAT. 75 (2), 2016 Offi cial Gazette of the Republic of Slovenia, 2010: Offi cial Ga- zette of the Republic of Slovenia 42/10. Retrieved May 28, 2014 from http://www.uradnili-st.si/1/objava.jsp?urlurid=201 02122 Pence, V. C., 2010: The possibilities and challenges of in vitro methods for plant conservation. Kew Bulletin 65, 593–547. Pence, V. C., 2011: Evaluating costs for the in vitro propagation and preservation of endangered plants. In Vitro Cellular and Developmental Biology – Plant 47, 176–187. Pierik, R. L. M., 1991: Commercial aspects of micropropagation. In: Prakash, J., Pierik, R. L. M., (ed.) Horticulture – new tech- nologies and applications, 141–153. Kluwer Academic Pub- lishers, Dordrecht. Rao, N. K., 2004: Plant genetic resources: Advancing conserva- tion and use through biotechnology. African Journal of Bio- technology 3, 136–145. Sarasan, V., Cripps, R., Ramsay, M. M., Atherton, C., Mcmichen, M., Prendergast, G., Rowntree, J. K., 2006: Conservation in vitro of threatened plants – Progress in the past decade. In Vi- tro Cellular and Developmental Biology – Plant 42, 206–214. Sharma, R. K., Wakhlu, A. K., 2001: Adventitious shoot regenera- tion from petiole explants of Heracleum candicans Wall. In Vitro Cellular and Developmental Biology – Plant 37, 794– 797. Sharma, R. K., Wakhlu, A. K., 2003: Regeneration of Heracleum candicans Wall plants from callus cultures through organo- genesis. Journal of Plant Biochemistry and Biotechnology 12, 71–72. Sharma, R. K., Wakhlu, A. K., Bolaria, M., 2004: Micropropaga- tion of Anethum graveolens L. through axillary shoot prolifer- ation. Journal of Plant Biochemistry and Biotechnology 13, 157–159. Šajna, N., Kušar, P., Slana-Novak, L., Novak, T., 2009: Notes on thermo- and hygropreference in Leiobunum roseum C. L. Koch, 1839 (Opiliones: Sclerosomatidae) in a habitat of Hlad- nikia pastinacifolia Reichenbach, 1831 (Spermatophyta: Api- aceae). Contributions to Natural History 12, 1111–1123. Šajna, N., Kušar, P., Slana-Novak, L., Novak, T., 2011: Benefi ts of low-intensive grazing: co-occurrence of umbelliferous plant (Hladnikia pastinacifolia Rchb.) and opilionid species (Pha- langium opilio L) in dry, calcareous grassland. Polish Journal of Ecology 59, 777–786. Šajna, N., Kavar, T., Šuštar-Vozlič, J., Kaligarič, M., 2012: Popu- lation genetics of the narrow endemic Hladnikia pastinacifo- lia Rchb. (Apiaceae) indicates survival in situ during the Pleistocene. Acta Biologica Cracoviensia Series Botanica 54, 84–96. Šajna, N., Šuštar-Vozlič, J., Kaligarič, M., 2014: New insight into the anatomy of an endemic Hladnikia pastinacifoia Rchb. Acta Biologica Cracoviensia Series Botanica 73, 19–28. Tavares, A. C. P., Salgueiro, L., Canhoto, J., 2009–2010: In vitro conservation of Angelica pachycarpa, an Iberian endemic Api- aceae of the Portuguese Berlanga Island. Revista del Jardin Botanico Nacional 30–31, 109–111. Tavares, A. C., Salgueiro, A. L., Canhoto, J., 2010a: In vitro prop- agation of the wild carrot Daucus carota L. subsp. halophilus (Brot.) A. Pujadas for conservation purposes. In Vitro Cellular and Developmental Biology – Plant 46, 47–56. Tavares, A. C., Salgueiro, A. L., Canhoto, J., Paiva, J., 2010b: Ibe- rian endemic Apiaceae: A reassessment for conservation pur- poses in Portugal. Studia Botanica 29, 13–37. Vittoz, P., Cherix, D., Gonseth, Y., Lubini, V., Maggini, R., Zbin- den, N., Zumbach, S., 2013: Climate change impacts on biodi- versity in Switzerland: A review. Journal for Nature Conserva- tion 21, 154–162. Wraber, T., 2009: The alpine fl ora. In: Bizjak, J. [texts], Lenarčič, M. [photos]: The Alps: a bird’s-eye view, 479–481. PanAlp, Nazarje.