70 ACTA BOT. CROAT. 77 (1), 2018 Acta Bot. Croat. 77 (1), 70–79, 2018 CODEN: ABCRA 25 DOI: 10.1515/botcro-2017-0011 ISSN 0365-0588 eISSN 1847-8476 Media composition affects seed dormancy, apical dominance and phenolic profile of Knautia sarajevensis (Dipsacaceae), Bosnian endemic Erna Karalija1,2, Sanja Ćavar Zeljković3,4, Petr Tarkowski3,4, Edina Muratović1, Adisa Parić1,2 1 University of Sarajevo, Faculty of Science, Department of Biology, Laboratory for Plant Physiology, Zmaja od Bosne 33–35, 71000 Sarajevo, Bosnia and Herzegovina 2 University of Sarajevo, Faculty of Science, Department of Biology, Laboratory for research and protection of endemic resources, Zmaja od Bosne 33–35, 71000 Sarajevo, Bosnia and Herzegovina 3 Centre of the Region Haná for Biotechnological and Agricultural Research, Central Laboratories and Research Support Faculty of Science, Palacky University, Šlechtitelů 27, 78371 Olomouc, Czech Republic 4 Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Genetic Resources for Vegetables, Medicinal and Special Plants, Crop Research Institute, Šlechtitelů 29, 78371 Olomouc, Czech Republic Abstract – Knautia sarajevensis is an endemic plant of the Dinaric Alps and is mainly distributed on Bosnian Mountains. Due to the quite large flower heads and easy maintenance, this plant has a potential use as a substi- tute ornamental plant for K. arvensis in perennial beds. The current study evaluated the germination process in different treatments in an attempt to suppress dormancy and increase germination rate, and to develop a suc- cessful protocol for micropropagation. An over 60% germination rate was achieved through cultivation of seeds on MS basal medium with reduced mineral nutrient composition and the absence of sucrose. On the other hand, a below 10% germination rate was achieved with untreated seeds. Suppression of apical dominance was achieved through application of high concentrations of kinetin, apical shoot decapitation or cultivation of shoots in liquid media. Overall, liquid cultures were more successful as a micropropagation system for this plant. Shoots spontaneously developed roots on multiplication treatments and were successfully acclimatized. Moreo- ver, phenolic compound profile was analysed in the light of the possible medicinal potential of this plant. Varia- ble amounts of total phenolic compounds as well as individual phenolics were recorded, according to treatment and solidification of media. An increase in rosmarinic acid content was reported for kinetin treatments and ac- climatized plants comparing to mother plants in natural habitat. The present study shows that choice of cyto- kinin concentration, explant type as well as culture type influences not only shoot proliferation and apical dom- inance suppression but also in vitro production of phenolics. Keywords: apical dominance, acclimatisation, endemic plant, Knautia sarajevensis, phenolics, seed dormancy * Corresponding author, e-mail: erna.k@pmf.unsa.ba; erna.karalija@gmail.com Introduction Knautia sarajevensis (Beck) Szabó is an endemic species of the Dinaric Alps that can be found in wood margins and woodland meadows in Bosnia. As an endemic plant species it is included in the Red List of flora for the Federation of Bosnia and Herzegovina and it is mainly found on moun- tains around Sarajevo (Mt. Igman, Bjelasnica, Trebevic, Ja- horina, Ozren; Đug et al. 2013). All of these mountains are subject to great anthropogenic impact due to tourism and to their role as popular recreational sites. These regions are also subject to constant deforestation and neglect, which affects habitat characteristics and plant life, especially plants like K. sarajevensis. Knautia sarajevensis is a clonal species and its reproduction is achieved through branching of vegetative organs, while seed germination and establishment of fully grown plants is considered to be rare and usually is linked to population establishment at new sites. Poor germination is probably an effect of seed dormancy, elaiosome presence and obligatory light induced germination (Mayer and Svoma 1998). Since K. arvensis is a popular ornamental plant used in perennial beds (Hartmann et al. 2010) K. sarajevensis too mailto:erna.k@pmf.unsa.ba mailto:erna.karalija@gmail.com MICROPROPAGATION PROCESS AFFECTS PHENOLIC PROFILE ACTA BOT. CROAT. 77 (1), 2018 71 must have a potential use as an ornamental and could re- place K. arvensis due to its larger flower heads and number of flowers than K. arvensis. Usually K. arvensis is propagated by dormant crown division or stem cuttings. In vitro culture research into the family Dipsacaceae is very scattered, and only micropropagation protocols for Scabiosa columbaria producing an average of 3.0 shoots per explant (Romeijn and Van Lammeren 1999) and S. caucasica (Hosoki and Nojima 2004) are available. Apical dominance is a term that signifies apical shoot growth and the inhibition of axillar shoot growth in perennial plants (Cline and Sadeski 2002), and can be a problem in the micropropagation of these spe- cies. In its natural habitat K. sarajevensis shows apical domi- nance of flowering over rosette formation and in the first year of growth, formation of a ground rosette takes place, while shoots bearing the flower heads are produced the following year. The reason for this probably lies in the importance of ac- cumulation of a critical mass for flowering, as already recorded for Succisa pratensis (Jongejans et al. 2006). Emergence of api- cal dominance in in vitro culture of K. sarajevensis is probably the result of a lack of critical mass accumulation and must be supressed in order to produce shoots in vitro. The potential medicinal properties of the Knautia ge- nus are still not well explored although there are data about Knautia arvensis plants as remedies for various skin disor- ders, and tea made from flowers and leaves of this plant can be used for many lung problems (Grieve 1931); this spe- cies is listed as a relaxant and blood purifier (Mattalia et al. 2013). Knautia bidens is listed as a rich source of phenolic compounds comparable to some Salvia species (Alali et al. 2007). Dipsacus genus has been broadly investigated for its medical properties, and there is evidence about its cytotox- ic and anticomplementary activities (Oh et al. 1999; Hung et al. 2005). Since Dipsacus and Knautia genera are closely related, they could share the same or similar medicinal po- tentials, so the importance of a micropropagation protocol development lies not only in the need for conservation but also in the potential medicinal uses of the genus Knautia. Accordingly, the aim of this study was to develop a success- ful micropropagation protocol, through plant regeneration from seeds, for conservation purposes. Also production of phenolic compounds was taken under consideration in rela- tion to concentrations of phenolics in mother plants. Materials and methods Aseptic seed germination Fully ripped fruits of Knautia sarajevensis were collected from a mother plant from a population located on Mt Igman. A voucher specimen of the seeds was marked and stored in the herbarium of the National Museum of Bosnia and Herze- govina (No. 51413). All fruits were cleaned, elaiosomes were removed as was epicalyx. Seeds were counted and separated into groups of 250 seeds for further treatments (Tab. 1). Pre- pared seeds were washed with 70% (v/v) ethyl alcohol for 30 sec, followed by 20 min submergence in 20% (v/v) so- lution of commercial sodium hypochlorite (4% active chlo- rine), then rinsed five times with sterile-distilled water. The experiment was conducted through a randomized design with 10 petri dishes containing 20 mL of media, and in each petri dish 25 seeds were inoculated for each treat- ment. Two sets of ten petri dishes for evaluation of germi- nation rate were used, one set for a 30 day, and other set for a 60 day period. All media were prepared according to Mu- rashige and Skoog (1962) (MS) basal salt composition, pH was adjusted to 5.8 prior to agar addition (0.8%) and were sterilised in an autoclave (1 bar, 121 °C, for 20 minutes). Su- crose (3.0%) was added before pH adjustment to all treat- ments except KS11 and KS12 (Tab. 1). Gibberellic acid (GA) was filter-sterilised and added after sterilisation of the media for appropriate treatments. All cultures were kept at 23 °C in growth chambers (70% humidity, 2000 lux light intensity, 16 h Tab. 1. Breaking dormancy and germination rate of Knautia sarajevensis seeds. MS – media composition according to Murashige and Skoog (1962), GA – gibberelic acid. Mean values not sharing the same letter(s) within one column are significantly different (P=0.01) according to Newman-Keuls test. Pre-treatment Treatment Incubation temperature % germination 30 days % germination 60 days KS1 – MS; 3% sucrose; 0 mg L–1 GA 23 °C 9.78±0.01h 24.51±0.00i KS2 – MS; 3% sucrose; 0.15 mg L–1 GA 23 °C 9.95±0.01h 31.94±0.00g KS3 30 days at +4 °C MS; 3% sucrose; 0 mg L–1 GA 23 °C 14.59±0.02g 41.15±0.00e KS4 30 days at +4 °C MS; 3% sucrose; 0.15 mg L–1 GA 23 °C 19.62±0.01e 61.13±0.01a KS5 30 days at +4 °C MS; 3% sucrose;0 mg L–1 GA 48 h +4 °C; +23 °C 30.15±0.00c 48.55±0.00c KS6 30 days at +4 °C MS; 3% sucrose; 0.15 mg L–1 GA 48 h +4 °C; +23 °C 33.05±0.01b 60.54±0.00b KS7 30 days at +4 °C; part of endosperm removed MS; 3% sucrose; 0 mg L–1 GA 23 °C 17.39±0.01f 40.61±0.00f KS8 30 days at +4 °C; part of endosperm removed MS; 3% sucrose; 0.15 mg L–1 GA 23 °C 19.14±0.00e 48.15±0.00d KS9 30 days at +4 °C; isolated embryo MS; 3% sucrose; 0 mg L–1 GA 23 °C 2.26±0.00 j 3.03±0.01l KS10 30 days at +4 °C; isolated embryo MS; 3% sucrose; 0.15 mg L–1 GA 23 °C 7.63±0.00i 7.90±0.00k KS11 – ½ MS; 0% sucrose 0 mg L–1 GA 23 °C 27.06±0.02d 48.20±0.00 j KS12 – ½ MS; 0% sucrose 0.15 mg L–1 GA 23 °C 65.60±0.01a 90.60±0.01h KARALIJA E., ĆAVAR ZELJKOVIĆ S., TARKOWSKI P., MURATOVIĆ E., PARIĆ A. 72 ACTA BOT. CROAT. 77 (1), 2018 photoperiod). Obtained seedlings were further used for the establishment of the K. sarajevenisis shoot cultures. Establishment of the shoot cultures and apical dominance suppression Roots were removed from all germinated seedlings prior to cultivation on shoot culture media. The obtained shoots were decapitated and apical and nodal parts were separately cultivated on media containing cytokinins (kinetin, 6-ben- zyladenin and zeatin respectively) and indole-3-butyric acid (IBA). Cytokinins were added in 3 different concentrations, i.e. 0.1, 1.0 and 10.0 mg L–1 alone or in combination with 0.1 mg L–1 of IBA. Treatments without plant growth regulators (PGR free) were used as control. Each treatment was rep- resented by 5 Erlenmeyer flasks in three independent rep- lications, and in each flask 5 explants were inoculated. All cultures were kept at 23 °C in a growth chamber (70% hu- midity, 2000 lux light intensity; 16 h photoperiod). After 21 days of cultivation the multiplication rate (number of reac- tive explants) (MR), multiplication index (average number of produced shoots per explant) (MI), presence of roots and presence of callus (Tab. 2) were recorded. Evaluation of the media consistency effect on apical dominance suppression In order to evaluate the media consistency effect on api- cal dominance suppression and biomass production, shoots about 5 cm in length with minimum of two pairs of leaves were cultivated in solid and liquid media with the addition of 0.1, 1.0 and 10.0 mg L–1 of kinetin (KIN). Treatment with- out PGR was used as control. All media contained 3.0% of sucrose, were prepared according to the MS procedure, and had 0.8% agar (only solid cultures). Media were sterilised in an autoclave, and all cultures were kept at 23 °C in a growth chamber (70% humidity, 2000 lux light intensity; 16 h photo- period). All treatments were incubated in a shaker (65 rpm), and comprised 5 Erlenmeyer flasks per treatment, with three independent replicates for all treatments. Every Erlenmeyer flask contained 50 mL of media and 5 explants (shoots; 75 per treatment). After 21 days of cultivation for all treatments the multiplication index, and the presence of roots and cal- lus were recorded. Plantlet acclimatisation During shoot multiplication, good rooting was achieved and no additional rooting was necessary. Plants with devel- oped roots were washed in tap water to remove the agar and then transferred into pots containing a mixture of soil and sand (3:2). All pots were kept under a 16 h photoperiod (2000 lux light intensity), and constant humidity (70%) and temper- ature (+23 °C). During the period of 15 days plants were grad- ually exposed to lower humidity (40%) and larger temperature fluctuation (±10 °C). After 20 days, the development of new leaves was noticed and plants were transferred in greenhouse. Spectrophotometric analysis of phenolic compounds Chemical analysis was done for mother plants and regen- erated in vitro plants as well as acclimatized plants (5 plants per sample). Phenolics were isolated from the aerial parts of plants by maceration in 80% methanol (HPLC grade) by in- cubation for 24 h at 4 °C. Extracts were centrifuged at 2000 rpm for 15 min, and supernatants were collected for further analysis. Total phenolic content was analysed according to Wolfe et al. (2003) using Folin-Ciocalteu reagent. Quantifi- cation was done according to calibration curve of gallic acid and expressed as mg of gallic acid equivalent per g of dry weight (mg GAE/g DW). Total flavonoid content was do- ne using the aluminium chloride method (Ordoñez et al. 2006). Concentration of flavonoids was estimated using the calibration curve of catechin. Results were expressed as mg of catechin equivalent per g of dry weight (mg CE/g DW). Total flavanol content was analysed according to modified method of Gadzovska et al. (2007) using 1% DMACA (w/v) reagent (p-dimethyl aminocinnamaldehyde in HCl:CH3OH, 8:92) and using calibration curve of catechin. Results were expressed as mg of catechin equivalent per g of dry weight (mg CE/g DW). Total proanthocyanidins content was anal- ysed using vanillin-HCl method (Wettstein et al. 1977) and Tab. 2. Effects of decapitation and kinetin application on the multiplication rate and index of Knautia sarajevensis cultivated in solid media. MR – multiplication rate (percentage of explants with formatted shoots), MI – multiplication index (average number of formatted shoots per explant), KIN – kinetin, IBA – indole-butyric acid. Mean values not sharing the same letter(s) within one column are significantly different (P=0.01) according to Newman-Keuls test. KIN (mg L–1) IBA (mg L–1) Nodal explants Apical explants MR MI Roots Callus MR MI Roots Callus 0.0 0.0 45.15 3.10±0.10c – – 0.00 1.00±0.00b – – 0.1 0.0 52.13 5.08±0.35a good, long – 0.00 1.00±0.00b small, under- developed – 0.1 0.1 43.15 2.80±0.33c good, long – 0.00 1.00±0.00b short – 1.0 0.0 42.34 2.40±0.07c short, thick – 0.00 1.00±0.00b short small, with indirect root regeneration 1.0 0.1 32.15 2.05±0.01c longer – 0.00 1.00±0.00b short 10.0 0.0 45.56 3.70±0.08b – – 1.00 2.00±0.05a – – 10.0 0.1 44.43 3.30±0.01c – – 0.00 1.00±0.00b – with shoot regeneration MICROPROPAGATION PROCESS AFFECTS PHENOLIC PROFILE ACTA BOT. CROAT. 77 (1), 2018 73 quantified according to the calibration curve of catechin. Re- sults were expressed as mg of catechin equivalent per g of dry weight (mg CE/g DW). Samples with the highest total phe- nolic content were further analysed by HPLC-UV technique. HPLC analysis of phenolic compounds Separation and analysis of flavonoids and phenolic ac- ids (hydroxycinnamic and hydroxybenzoic acids) were per- formed on a Shimadzu LC-2010c HPLC using Phenomenex Kinetex C18 (2.6 µm ID, 150 × 4.6 mm) column. The mobile phase included component A (20 mM formic acid in water) and component B (acetonitrile) in a defined gradient (0 min 5% B; 4 min 5% B; 54 min 40% B; 60 min 40% B; 60, 5 min 5% B; 70 min 5% B; 70 min stop) at flow rate 0.4 mL/min. HPLC was equipped with UV detector, and absorbance was monitored at 270 nm. Concentration of flavonoid compo- nents was calculated in relation to calibration curves of stan- dards: myricetin, quercetin, naringenin, apigenin, kaemph- erol, chrisin, pinocembrin and galangin. For phenolic acids standards of chlorogenic, caffeic, sinapic, ferulic, rosmarinic, gallic, 4-hydroxybenzoic, vanillic, syringic and salicylic ac- ids were used (concentration range: 1×10–7 mol L–1 – 1×10–3 mol L–1). All standards used were purchased from Sigma and were of at least analytical grade. Statistical analysis All results were expressed as the mean values (±stan- dard deviation; STDEV) of the three independent replicates. Analysis of variance of parametric data was done accord- ing to ANOVA test using Newman-Keuls test as a post hoc analysis. Differences between treatments were evaluated at p<0.01. Correlation coefficient was calculated according to Pearson product-moment correlation coefficient at p<0.05. All statistical testing was done using Statistica 10.0 software (Copyright© StatSoft. Inc. 1984–2011). Results Aseptic seed germination Seeds of K. sarajevensis have elaiosomes and the remov- al of elaiosomes and seed coat was necessary for dormancy suppression. Isolation of embryos, one of the methods for dormancy suppression, was also conducted. Different cold pre-treatments for elaiosome-free seeds were used for im- provement of the germination rate in combination with gib- berellic acid application. The efficiency of gibberellin stimu- lation of seed germination in K. sarajevensis depended upon the pre-treatment and cultivation conditions. Seedlings de- veloped a pair of leaves 15 days after cultivation but germina- tion time was unsynchronized within one treatment. When no cold pre-treatment was applied maximum germination rate was prolonged up to 2 months even with GA applica- tion. Incubation of non-imbibed seeds at +4 °C for 30 days increased germination percentage. Further incubation at 4 °C after imbibition of cold pre-treated seeds (KS5 and KS6 treatments) significantly increased seed germination espe- cially in combination with GA application and maximum germination rate was achieved in the first 30 days (Tab. 1). Removal of sucrose in combination with reduction of basal salts in the media significantly induced germination and re- duced duration of germination period, especially when GA was applied (Tab. 1), moreover up to 65% of the seeds ger- minated in first 30 days. Establishment of the shoot cultures and apical dominance suppression In preliminary research (data not shown), seedlings were cultivated on media containing 0.1, 1.0 and 10.0 mg L–1 BA (6-benzyladenine), ZEA (zeatin) and kinetin (KIN) in solid media. During cultivation, emergence of apical dominance was noticed and no shoot formation was recorded. Shoots cultivated on solid media containing BA and ZEA showed signs of chlorosis and root formation was very low, while kinetin induced good root formation, and no chlorosis was noted but apical dominance was present. For further multi- plication and apical dominance suppression seedlings from which apical and nodal explants were derived were used. Apical parts of the shoots and nodal segments were separate- ly cultivated on media containing different concentrations of kinetin alone or in combination with 0.1 mg L–1 IBA. Culti- vation of explants in kinetin-containing media provided dif- ferent responses depending upon kinetin concentration and explant type. Apical explants did not produce any shoots ex- cept when the highest concentration of KIN was applied (10 mg L–1), while nodal explants produced shoots in different frequencies. The highest multiplication rate and index were reported for treatment with 0.1 mg L–1 of KIN using nodal explants (Tab. 2, Fig. 1). High concentrations of KIN in this study also induced callus formation in the basis of nodal ex- plants with indirect shoot regeneration (Tab. 2, Fig. 1). Addi- tion of IBA to the media had inhibitory effects on shoot and root formation (Tab. 2, Fig. 2). Plants with good roots were acclimatized in greenhouse conditions with a survival rate over 70%. All plants formed good ground rosettes compris- ing up to 10 leaves with no shoot formation demonstrating a 2 year vegetation period as recorded in the natural habitat. Evaluation of liquid media effect on apical dominance suppression In solid culture shoots were short (5–7 cm) and no elon- gation was noticed and apical dominance suppression was only possible by removal of shoot apex or by application of high kinetin concentrations. In contrast, the highest concen- tration of KIN (10 mg L–1) was unfavourable in liquid sys- tem and plants decayed within 3 days and this treatment was not further analysed. Shoots cultivated in the liquid media containing 0.1 mg L–1 of KIN demonstrated a 100% multipli- cation rate and multiplication was achieved mainly through proliferation of several axillary buds (Fig. 3A). Also, develop- ment of elongated shoots was reported (Fig. 3B). Apical dom- inance was suppressed and production of up to 5.4 shoots per explant was achieved using a low concentration of kinetin. KARALIJA E., ĆAVAR ZELJKOVIĆ S., TARKOWSKI P., MURATOVIĆ E., PARIĆ A. 74 ACTA BOT. CROAT. 77 (1), 2018 Fig. 1. Effects of decapitation and kinetin application on apical dominance suppression in Knautia sarajevensis; nodal explants cultivated in solid media containing kinetin: (A) 0.1 mg L–1, (B) 1 mg L–1, (C) 10 mg L–1, D) 0 mg L–1; apical explants cultivated in media containing kinetin: (E) 0.1 mg L–1, (F) 1 mg L–1, (G) 10 mg L–1, (H) 0 mg L–1. Bars = 1 cm. Fig. 2. Effects of decapitation and kinetin (KIN) application in combination with indole-3-butyric acid (IBA) on apical dominance sup- pression in Knautia sarajevensis; nodal explants cultivated in solid media containing: (A) 0.1 mg L–1 KIN and 0.1 mg L–1 IBA, (B) 1 mg L–1 KIN and 0.1 mg L–1 IBA, (C) 10 mg L–1 KIN and 0.1 mg L–1 IBA, (D) 0 mg L–1 KIN and 0 mg L–1 IBA; apical explants cultivated in solid media containing: (E) 0.1 mg L–1 KIN and 0.1 mg L–1 IBA, (F) 1 mg L–1 KIN and 0.1 mg L–1 IBA, (G) 10 mg L–1 KIN and 0.1 mg L–1 IBA, (H) 0 mg L–1 KIN and 0 mg L–1 IBA. Bars = 1 cm. MICROPROPAGATION PROCESS AFFECTS PHENOLIC PROFILE ACTA BOT. CROAT. 77 (1), 2018 75 Higher concentrations of KIN (1 mg L–1 and 10 mg L–1) had negative effects on shoot proliferation and induced vitri- fication of the plants. In this study the use of a small amount of the medium (50 mL) and a low kinetin concentration were enough for the avoidance of vitrification during mul- tiplication (Fig. 3C). Moreover, developing shoots were not submerged in the media because explants floated due to the large leaves and the hairs on the leaves in which air can be trapped. Phenolic compounds profile Analysis of total phenolic content in K. sarajevensis showed a high concentration of phenols and flavonoids in the aerial part of mother plants (Tab. 3). HPLC analysis showed that around 30% of phenolic profile is composed of phenolic acids (gallic, 4-hydroxybenzoic, vanillic, salicylic, chlorogenic, caffeic, sinapic, ferulic and rosmarinic acids), (Tab. 4) and some flavonoids: myricetin as a most abundant (2.72 nmol mg–1), and apigenin and kaempherol in traces. Similar profile with lower concentration of individual com- ponents was found in in vitro cultivated plants as well as in acclimatized plants. Elevation of rosmarinic acid concen- Tab. 3. Phenolic contents in Knautia sarajevensis shoots after cultivation in solid and liquid media. K – kinetin, 01 – 0.1 mg L–1, 1 – 1.0 mg L–1, S – solid culture, L – liquid culture, PGR– no plant growth regulators added, MP – mother plant, AP – acclimatised plant. Mean values not sharing the same letter(s) within one column are significantly different (P=0.01) according to Newman-Keuls test. Treatment Phenolics (mg g–1 DW–1) Total phenols Total flavonoids Flavanols Proanthocyanidins MP 121.04±8.97a 154.51±16.11a 6.19±0.03a 33.46±3.86a AP 33.16±8.00c 165.13±4.38a 0.94±0.00cb 21.50±0.77b PGR S 31.14±3.68cd 63.27±11.71b 0.83±0.12cd 2.09±0.04e PGR L 21.98±0.15e 27.86±1.70cd 0.53±0.11cd 14.63±0.07c 01K S 20.31±3.55e 25.57±2.48cd 0.31±0.01d 3.46±0.04e 01K L 24.488±3.62de 34.24±4.84cd 0.80±0.01cd 9.40±0.42d 1K S 26.02±2.52d 33.88±3.18cd 0.46±0.02cd 3.73±0.02e 1K L 56.50±2.67b 49.24±4.02bc 1.51±0.03b 4.16±0.46e tration was reported for acclimatised plants and some KIN treatments (Tab. 4); in addition, the synthesis of syringic acid under KIN treatment was noticed. Discussion Natural populations of endemic Knautia sarajevensis are subject to anthropogenic impacts and it is important to develop a micropropagation protocol in order to give op- portunities for the growth of this species in new habitats. Micropropagation of endangered species enables plant prop- agation regardless of the season from a small number of mother plants (Mikulík 1999). For conservation purposes it is recommended to use seed for establishment of the cul- tures as they have a broader genetic base (Chua and Hen- shaw 1999). Removal of elaiosomes alone was not sufficient for dor- mancy removal in K. sarajevensis seeds, and different cold pre-treatments combined with gibberellic acid application were used for improvement of the germination rate. Gibber- ellic acid alone or in combination with cold treatment can break dormancy and promote germination as recorded for Fig. 3. Apical dominance suppression and multiplication of Knautia sarajevensis shoots in liquid culture: A) bud clusters, B) shoot multi- plication, and C) liquid media cultivation. Bars = 1 cm. KARALIJA E., ĆAVAR ZELJKOVIĆ S., TARKOWSKI P., MURATOVIĆ E., PARIĆ A. 76 ACTA BOT. CROAT. 77 (1), 2018 Tab. 4. HPLC analysis of phenolic compounds in Knautia sarajevensis shoots cultivated in solid and liquid media. K – kinetin, 1 – 1.0 mg L–1, S – solid culture, L – liquid culture, PGR– no plant growth regulators added, MP – mother plant, AP – acclimatised plant, nd – not detected. Hydroxybenzoic acid (nmol mg–1 of extract) Treatment MP AP PGR S PGR L 1K S 1K L Gallic acid 8.71 4.75 2.88 1.23 6.15 nd 4-Hydroxybenzoic acid 46.05 11.07 14.40 7.16 16.41 2.72 Vanillic acid 0.38 0.62 0.12 0.37 4.87 1.35 Syringic acid nd 0.36 nd 0.19 3.09 0.94 Salicylic acid 20.59 11.40 12.75 7.19 18.97 4.18 Chlorogenic acid 1.67 0.18 1.30 0.43 0.32 2.49 Caffeic acid 0.031 0.59 0.32 0.35 0.20 nd Sinapic acid 53.65 1.98 0.61 0.63 0.95 0.10 Ferulic acid 57.69 2.59 0.56 nd 0.94 0.27 Rosmarinic acid 2.61 12.71 13.86 5.01 11.66 2.61 Myricetin 2.72 0.47 nd 0.80 0.77 nd SUM 194.11 45.89 46.81 23.39 64.27 14.66 many different genera (Nau 1996, Raeber and Lee 1991). Ef- ficiency of gibberellin stimulation of seed germination in K. sarajevensis depended upon the pre-treatment and cultiva- tion conditions used, but was usually pronounced when im- bibed seeds were additionally exposed to cold treatment as previously recorded for other species (Yamauchi et al. 2004, Okamoto et al. 2006). Endosperm plays an important role in seed germination (Yan et al. 2014), synthesis of hydrolytic enzymes, sensing the red and far-red light necessary for ger- mination (Lee et al. 2012) and controlling embryo growth (Lee et al. 2010). Removal of part of the endosperm or isola- tion of embryos can be a useful tool for seed dormancy re- moval (Yan et al. 2012) since seed dormancy can be caused through an impermeable seed coat or other dormancy fac- tors from the endosperm (Nonogaki, 2014). In this study such treatments did not stimulate seed germination in K. sarajevensis even when GA was applied, as compared to oth- er treatments. An explanation for this could lie in the com- position of K. sarajevensis endosperm. Seeds of the Knau- tia genus contain oils as a reserve of nutrients (Mayer and Svoma 1998, Tonguç and Erbaş 2012). For germination and energy gain, oils from the endosperm must be catabolised though gluconeogenesis in order to be passed to the embryo in the form of sucrose (Penfield et al. 2004). Endosperm re- moval excludes the reserve oils in which case embryos are under stress since they have no energy source. It is record- ed that embryos in suboptimal growth conditions experi- ence restriction of the gluconeogenic pathway (Rylott et al. 2003), which could lead to reduction in the growth and ger- mination rate. Removal of sucrose in combination with a re- duction of basal salts in the media was the most successful treatment for elaiosome-free seeds of K. sarajevensis. Pres- ence of sucrose in media can induce osmotic stress and in- hibit seed germination. Also, growth of the seedlings can be inhibited by sucrose hydrolytic products, which are formed during media sterilisation process (Sawyer and Hsiao 1992, Pan and van Staden 1999). Elevation of germination rate by reduction of media mineral composition is recorded also for other species as well (Mishra et al. 2013). Apical dominance of ground rosette over flowering stems is present in natural habitats of K. sarajevensis, and this phe- nomenon has previously been recorded for Succisa pratensis (Jongejans et al. 2006), and it is probably the reason for the recorded apical dominance emergence in shoot cultures in this study. Choice of an adequate cytokinin and a concen- tration that stimulates shoot induction and elongation var- ies depending upon plant species and must be optimised for each particular plant species (Jana et al. 2013). Successful multiplication was achieved by low kinetin concentrations and such a treatment has been recorded for a relatively small number of species (Deo et al. 2014). Elevation of kinetin concentration induced callus formation, which has been re- ported in Scrophularia takesimensis cultures (Ding and Chen 2007), with the possibility for regeneration from petiole and leaf (Wang et al. 2013); similar results were also reported for other Scabiosa species (Hosoki and Nojima 2004). Decapita- tion was relatively successful as a tool for apical dominance suppression in K. sarajevensis shoot cultures. Apical shoot removal was previously recorded as an effective method for apical dominance suppression (Podwyszynska 1997). Also it is considered that the endogenous auxin to cytokinin ratio suppresses bud outgrowth and an exogenous supply of cyto- kinins can change this ratio (Hillman 1984), which in turn stimulates bud outgrowth. Senescence of older leaves was observed for all apical explants and control treatment (no plant growth regulators added), which probably impaired successful multiplication (Mensual-Sodi et al. 2007). Kine- tin application reduced senescence in nodal explants, and the role of kinetin in senescence has been previously dem- onstrated (Mukharjee and Kumar 2007). Negative effects of exogenous auxins on multiplication have been previously re- ported. Single shoot development and callus induction was demonstrated due to apical dominance resulting from in- creased auxin concentration (Buah et al. 2010). In this study MICROPROPAGATION PROCESS AFFECTS PHENOLIC PROFILE ACTA BOT. CROAT. 77 (1), 2018 77 high endogenous auxin levels were responsible for root for- mation on media containing only kinetin while additional exogenous auxins had a negative effect on rhizogenesis. The negative effect of high auxin concentration on root forma- tion has been well documented with reference to a number of species (Buah et al. 2010, North et al. 2010). High survival rate during acclimatisation has been reported for some Sca- biosa species (Hosoki and Nojima 2004, Wang et al. 2013), but data regarding Knautia species are scattered. Since suppression of apical dominance is possible through the use of liquid cultures (Mehrotra et al. 2007), in the next step we used a liquid culture using the same kinetin concentrations that were favourable for multiplication in a solid culture with no signs of callus formation to ensure that only shoots by direct regeneration were obtained. Develop- ment of bud clusters and mechanical separation of shoots in liquid systems provide an efficient delivery system in micro- propagation (Levin et al. 1997, Ziv et al. 1998). Production of buds enables the production of a large number of plants (Takayama and Misawa 1981). The contact of the explants with the medium facilitates the uptake of nutrients and plant growth regulators, which lead to promotion of shoot and root formation (Sandal et al. 2001). Forced aeration, due to continuous shaking of the medium, provides oxygen supply to the whole explant, which stimulates growth (Mehrotra et al. 2007). There are many studies suggesting that agar elimi- nation causes vitrification of tissue during micropropagation (John 1986, Kevers et al. 1987). The problem of asphyxia- tion is a common problem in liquid cultures (Mehrotra et al. 2007). In this study reduction of the media amount and low kinetin concentration were enough for avoidance of vitrifi- cation during multiplication. Moreover, developing shoots were not submerged in the media because explants tended to float due to their large leaves and the hairs on the leaves among which air can be trapped. The use of surface tension and floating properties of an explant is very useful for the avoidance of submergence of tissues and vitrification phe- nomena in a liquid medium (Debergh et al. 1992). Since closely related members of the Dipsacaceae family have been demonstrated to have medicinal properteis (Oh et al. 1999, Hung et al. 2005, Mattalia et al. 2013), prospects for the micropropagation of K. sarajevensis as a potential medicinal plant also included phytochemical screening as the first step towards such a use of the plant. There are no available data about the phenolic compounds found in K. sarajevensis, and also very little is known about the genus Knautia, in general; some of the identified components are reported for the first time. According to the available da- ta apigenin, swertijaponin, and giganteoside A were found in K. montana (Movsumov et al. 2011); cryptochlorogenic and chlorogenic acid and isovitexin 7-β-D-glucopyranoside were detected in K. arvensis (Moldoch et al. 2011), while a high concentration of polyphenolics in K. bidens has been reported and the plant is considered a rich source of phe- nolic compounds (Alali et al. 2007). Kinetin is considered to be a promoter of secondary metabolite synthesis (Klessig and Malamy 1994, Kim et al. 2009), as demonstrated in this study. There is less synthesis than in the mother plants, but some components that are not recorded for mother plants have been identified in kinetin-treated in vitro plants, which is in accordance with other studies (Rao and Ravishankar 2002, Luczkiewicz and Gold 2005). Manipulation of PGR concentration and culture conditions can provide changes in secondary metabolites (Rao and Ravishankar 2002, Luczkie- wicz and Gold 2005, Lucchesini et al. 2009) as also shown in this study. Use of shoots instead of callus is more convenient and it is also considered that more differentiated tissues pro- duce more metabolites (Luczkiewicz and Gold 2005, Nath and Buragohain 2005, Sood and Chauhan 2010), and use of liquid systems gives better results (Savio et al. 2011) as dem- onstrated in this study. To conclude, this study provides a successful microprop- agation protocol for Knautia sarajevensis and accordingly a useful tool in the establishment of new population sites as a method for conservation of this endemic species and for its potential use as an ornamental plant, replacing K. arvenisis in perennial beds. Successful micropropagation can be per- formed by germination of seeds on media without sucrose and reduced basal salt concentration. Apical dominance can be overcome by decapitation of shoots or cultivation in liq- uid media. Due to the high endogenous concentration of auxin, rooting media is not necessary and roots develop in multiplication media with successful acclimatization of root- ed plants. Elevation of secondary metabolite concentration (compared to control) due to kinetin application in solid cul- tures gives an input for the possible elicitation of phenolic acids in this plant. 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