In vitro direct organogenesis of the medicinal single-mountain local prioritized vulnerable Greek endemic Achillea occulta under different medium variants

Achillea species are of high medicinal value based on their use in traditional medicine and their content in secondary metabolites with potential application in dermatology and cosmetic industry. Achillea occulta is a single-mountain local prioritized vulnerable Greek endemic encountering propagation problems through seeds and root division. In vitro propagation can be used for the rapid and mass production of clonal plants complying with phytochemical and sanitary quality criteria. In this study, several culture medium variants were tested including the effect of different indole-3-butyric acid (IBA) concentrations with 6-benzyladenine (BA), different cytokinin types, agar concentrations, and zeatin (ZT) sterilization methods on in vitro shoot proliferation, as well as the effect of different auxin types and concentrations on in vitro rooting and ex vitro survival rate of shoot-tip explants. The results showed BA was the most preferable cytokinin type as evidenced by all proliferation attributes and autoclaved ZT provided longer shoots. Therefore, the two more promising and cost-effective treatments for proliferation were 2.2 μM BA + 0.25 μM IBA or 4.4 μM BA + 0.5 μM IBA after 21 days of culture on modified Murashige-Skoog (MS) medium (x2 Fe) enriched with 20 g L sucrose and 6-7 g L Agar (90-93.8% shoot formation, 2.7-2.9 shoots 0.4-0.6 cm long, 2.5-2.6 proliferation rates). Explants rooted better after 21 days of culture on plain MS medium with 2 μM IBA or 1 μM α-naphthalene acetic acid (NAA) (13.3-20% rooting, 1-1.5 roots 1.8-2 cm long). NAA gave more roots, IBA longer roots, while both auxins exhibited highest rooting (13.33-20%) and ex vitro survival rates (50-66.7%).


Introduction Introduction Introduction Introduction
Plant species that belong to the genus Achillea (Asteraceae) have been reported to be of high medicinal value based on their use in traditional medicine as a natural remedy for the healing of several ailments including headache, pains, wounds, flatulence, bleedings, inflammation, dyspepsia, and spasmodic diseases 2 (Mohammadhosseini et al., 2017). The medicinal properties within Achillea species are attributed to their content in a wide variety of secondary metabolites such as flavonoids, phenolic acids, terpenes, phytosterols, organic acids, fatty acids, and alcohols with potential use in dermatology and cosmetic industry as rich sources of active phytochemicals (Strzępek-Gomółka et al., 2021). More than 130 Achillea species are wild perennial herbs, the majority distributed mainly in Northeastern Europe and Northwestern Asia, exhibiting antimicrobial, antioxidant, and anti-inflammatory action (Stojanovic et al., 2005).
According to Constantinidis and Kalpoutzakis (2009), Achillea occulta is a single-mountain local endemic of the Greek flora with conservation priority as a threatened one due to fires and overgrazing. Based on IUCN criteria (2001), this species has been characterized as 'vulnerable' (Constantinidis and Kalpoutzakis, 2009) and is not protected by the Hellenic Presidential Decree 67/81 (Dimopoulos et al., 2016). The natural habitat of this plant includes cliffs, rocks, walls, ravines, and boulders in Mt Koulochera and Komboriza, SE Peloponnese, Greece, at 700-1.126 m (Constantinidis and Kalpoutzakis, 2009).
Seed germination and asexual root division are traditional propagation methods used for Achillea spp. also commonly known as yarrows but in a very restricting time period i.e., a particular month of the year (Chaturvedi et al., 2007). Mass production of pathogen-free and genetically uniform plant material of yarrow on a commercial scale both qualitatively and quantitatively, and throughout the whole year can be achieved through plant tissue culture strategies including micropropagation for standardized formulations in the chemical constituents of the plants and their parts, fulfilling the ever-increasing demand of uniform plant-based medicines warrants (Chaturvedi et al., 2007). New methods derived from advances in biotechnology could contribute more efficiently to the conservation of plant diversity and its subsequent evaluation process (Manole-Păunescu, 2014). In addition, new methodological biotechnology tools can reinforce in situ measures and ex situ classical strategies related to the collection, multiplication, and conservation of plant biodiversity (Holobiuc et al., 2018). Up to now, there is a limited number of research studies regarding in vitro regeneration of Achillea species including calli and cell suspension cultures of A. millefolium (Figueiredo and Pais, 1991), in vitro propagation of A. ptarmica (Cellarova et al., 1982), micropropagation of A. asplenifolia (Wawrosch et al., 1994) and A. occulta (Grigoriadou et al., 2011), callus induction of A. biebersteinii from leaf and petiole explants (Kahrizi and Kakaei, 2010).
This study was undertaken with the objective to develop an efficient in vitro protocol that would maintain and propagate A. occulta, an important species by testing several factors in an attempt to optimize the explants response. Several parameters were taken into consideration for the in vitro shoot proliferation stage of A. occulta including the effect of (1) different indole-3-butyric acid (IBA) concentrations combined with 6benzyladenine (BA), (2) different concentrations of the gelling agent "agar", (3) different cytokinin types and (4) different sterilization methods of the cytokinin "zeatin (ZT)" either autoclave-sterilized (no filter used) or filter-sterilized without undergo autoclaving, applied to the culture medium alone and in combination with different BA concentrations. In the following stages, different auxin types and concentrations were tested in order to determine the ideal auxin type and its optimum concentration for maximal in vitro rooting performance and better adaptation of the rooted microshoots to ex vitro greenhouse conditions through a gradual acclimatization and hardening process.

Plant material and culture conditions
Shoot-tip explants, 0.5 cm long, derived from previous in vitro cultures sub-cultured every four weeks on ΜS (Murashige and Skoog, 1962) (Duchefa Biochemie, The Netherlands) basal medium containing 1.32 μM 6-benzyladenine (BA) (Duchefa Biochemie, The Netherlands), 0.15 μM indole-3-butyric acid 3 (ΙBA)(Duchefa Biochemie, The Netherlands), 20 g L -1 sucrose (Duchefa Biochemie, The Netherlands) and 6.5 g L -1 agar (Sigma-Aldrich) were used as the experimental plant material in all five experiments conducted, the first four related to in vitro shoot proliferation, and the fifth and last one regarding in vitro rooting. The pH value of the culture media was adjusted to 5.8 before adding the gelling agent and prior to autoclaving at 121 °C for 20 min. The explants were transferred into Magenta TM vessels (Baby food jars, autoclavable, reusable, 62.4 × 95.8 mm, size: 200 mL, Sigma-Aldrich, owned by Merck KGaA, Darmstadt, Germany), each containing 35 mL of the culture media as described below. Magenta TM B-caps were used for covering the vessels. The cultures were maintained in a growth chamber under 16h light/ 8h darkness photoperiod regime, light intensity of 40 μmol m -2 s -1 supplied by cool white fluorescent lamps, and temperature of 22 ± 1 o C. The same controlled environmental conditions (photoperiod, illumination, temperature) were followed for all experiments carried out and described below during the in vitro cultures of A. occulta explants inside the growth chamber.
In vitro shoot proliferation Different IBA concentrations combined with BA Five different concentrations of the auxin IBA (0, 0.5, 1.25, 2.5 and 5 μM) were tested. The basal culture medium used was the modified MS [double amount of Fe (x2 Fe)] enriched with 20 g L -1 sucrose, 4.4 μM BA and solidified with 6 g L -1 Plant Agar (Duchefa Biochemie). After 21 days of culture, the following measurements were recorded: percentage of shoot formation (%), number of shoots per explant, shoot length (cm) and proliferation rate. The experiment included 5 treatments with 16 repetitions (explants)/ treatment (4 explants/ vessel x 4 vessels/ treatment).

Different agar concentrations
Three different concentrations of the gelling agent "agar" (Sigma-Aldrich) (6, 7 and 8 g L -1 ) were tested. The culture medium used was the modified MS (x2 Fe) enriched with 20 g L -1 sucrose, 2.2 μM BA and 0.25 μM IBA. After 21 days of culture, the following measurements were recorded: percentage of shoot formation (%), number of shoots per explant, shoot length (cm) and proliferation rate. The experiment included 3 treatments with 20 repetitions/ treatment (4 explants/ vessel x 5 vessels/ treatment).

Different cytokinin types
Four different cytokinin types, i.e. BA, 2-isopentenyl-adenine (2-ip), zeatin (ZT) and kinetin (KIN), each applied in a 4.4 μM concentration were tested. The culture medium used was the modified MS (x2 Fe) enriched with 20 g L -1 sucrose, 0.44 μM IBA and solidified with 7 g L -1 Plant Agar. After 21 days of culture, the following data were recorded: percentage of shoot formation (%), number of shoots per explant, shoot length (cm) and proliferation rate. The experiment included 5 treatments with 20 repetitions/ treatment (4 explants/ vessel x 5 vessels/ treatment).
ZT application method and BA concentration ZT applied either autoclave sterilized (no-filter used) or filter-sterilized without autoclaving in a concentration of 4.4 μM. The individual effect of BA applied at 4.4 μM as well as the combined effect of 4.4 μM ZT (non-filtered, filtered) with 4.4 μM BA were tested. Therefore, the experiment included five treatments, i.e. 4.4 μM BA, 4.4 μM ZT (non-filtered), 4.4 μM ZT (filtered), 4.4 μM ZT (non-filtered) + 4.4 μM BA, and 4.4 μM ZT (filtered) + 4.4 μM BA. The culture medium used was the modified MS (x2 Fe) enriched with 20 g L -1 sucrose, 0.44 μM IBA and solidified with 7 g L -1 Plant Agar. After 21 days of culture, the following data were recorded: percentage of shoot formation (%), number of shoots per explant, shoot length (cm) and proliferation rate. The experiment included 5 treatments with 20 repetitions/ treatment (4 explants/ vessel x 5 vessels/ treatment).

Ex vitro acclimatization, hardening and survival of rooted microshoots
During early spring, rooted microshoots were rinsed with tap water to remove the adhering medium and planted in multi-hole propagation trays (100 mL volume per plantlet) containing a substrate mixture composed of peat (Terrahum) and perlite (1:1 v/v). The trays were transferred to a heated mist system in the greenhouse [at 18-21 o C base temperature, 15-25 o C air temperature and 70-85% relative humidity (RH)] for 30 days under reduced light intensity conditions (thermal curtains). Following that, the plants were successively transplanted in larger pots (0.33 L and 2.5 L) containing a peat moss (TS2): perlite: soil (2: 2: 1/2 v/v) substrate, and then placed in the greenhouse bench under increased light intensity and gradual RH decrease (5% per day) conditions. In early summer the plants transferred outside the greenhouse under a 50% shading net and automated sprinkling irrigation system.

Statistical analysis
The experimental layout was completely randomized. The means were subjected to analysis of variance (ANOVA) and compared using the Duncan multiple-range test (p ≤ 0.05). The first, second and third in vitro shoot proliferation experiments were not multi-factorial and means were subjected to one-way ANOVA. The fourth shoot proliferation experiment was a 2×2 factorial one with two different application methods of ZT (non-filtered-autoclave-sterilized, filtered-sterilized without autoclaving) and two BA concentrations (0 and 4.4 μM). The main effect of factors (ZT application method, BA concentration) and their interaction was determined by General Linear Model/ 2-way ANOVA). The rooting experiment was a 3×4×4 factorial one with three auxin types (IBA, NAA, IBA + NAA), four IBA concentrations (0, 1, 2 and 5 μM), and four NAA concentrations (0, 1, 2 and 5 μM). The main effect of factors (auxin type, IBA concentration, NAA concentration) and their interactions were determined by General Linear Model/ 3-way ANOVA.

Treatments
Shoot formation (%)  Different cytokinin types In the case of the control containing 0.44 μM IBA (cytokinin-free), only the 5% of the explants resulted in multiple shoot formation. Similar levels in higher shoot formation percentages (60-85%) were recorded in the other treatments containing different cytokinin types, differing statistically significant as compared with the control (5%) ( Table 3; Figures 3A-E). All four culture media enriched with different cytokinin types (BA, 2-ip, ZT, KIN) led to a significant increase in shoot numbers (1.85-2.55) and proliferation rates (0.96-1.66) in relation to the control (1.05 shoots/ explant with a 0.49 proliferation rate). Between BA and KIN, BA was more beneficial in raising shoot number. Concerning shoot length, ZT was the preferred cytokinin type as compared to KIN, whereas the other three treatments i.e control, BA + IBA, and 2-ip + IBA exhibited similar non-significant values with either ZT + IBA or KIN + IBA. Among the four cytokinins, BA was the most effective in terms of shoot formation % than the other three cytokinins (2-ip, ZT, KIN) presenting nonsignificant differences. In particular, shoot number (2.55) and proliferation rate (1.66) were maximum under BA + IBA treatment, while shoot length was the largest (0.70 cm) in the combined effect ZT + IBA. The absolute highest value for shoot formation (85%) was achieved by adding BA + IBA to the culture medium. Therefore, taking simultaneously all shoot multiplication parameters into consideration, BA was the most effective cytokinin type (Table 3).  7   Table 3. Table 3. Table 3.  Figure 3. Figure 3. Figure 3. ZT application method and BA concentration BA, or ZT (autoclave sterilized or filter-sterilized without autoclaving) applied either alone or in combination with BA gave 60-85% shoot formation percentages and 2.00-2.55 shoots/ explant without significant differences. Shoot length was significantly higher (0.70 cm) in autoclave-sterilized ZT application, while proliferation rates were significantly higher when BA used alone (1.66 cm) or when filter-sterilized ZT combined with BA (1.51 cm) (Table 4). Table  Table Table  Table 4

9
(A) (B) (C) (D) Figure 4. Figure 4. Figure 4. In vitro shoot proliferation Different IBA concentrations combined with BA The concentration of macronutrients in the MS culture medium is 3-to 4-fold higher than in other media tested (Murashige and Skoog, 1962). In this study, the MS medium was used as the basal one but modified on the concentration of chelate Fe which was doubled. According to Monteiro et al. (2000), there are several factors in tissue culture systems such as genotype, temperature, pH, and plant growth regulators (PGRs), among others that affect the uptake, transport or requirement of Fe, leading to decreased leaf chlorophyll content and phenomena of chlorosis. The reason for the use of the chelate form and double concentration of Fe in the MS basal medium [FeNaEDTA (x2)] in this study with A. occulta can be attributed to the fact that Fe becomes less exchangeable and available to plants as compared with the chelate form of Fe, especially under the lower temperatures used for incubation of tissue cultures inside growth chambers (Grigoriadou et al., 2011). The use of different categories of PGRs mainly cytokinins and auxins, different types of cytokinins (i.e., BA, 2-ip, ZT, KIN) or auxins (IBA, NAA, IAA, 2,4-D) and their ratio as incorporated into the culture medium play a critical role in the established type of culture (Păunescu, 2009). In the present study, 4.4 μM BA combined with the lowest applied IBA concentration of 0.5 μM proved to be the most effective cytokinin/auxin ratio (9:1) regarding proliferation rate (2.52), while BA along with higher IBA concentrations (1.25-5 μM) gave non-significant proliferation rates as compared with the control (IBA-free). In consistency with our findings, shoot proliferation rate is reported to be enhanced under combinations of higher cytokinin to lower auxin concentrations (Biswas et al., 2011), possibly due to higher level of degradative metabolites in tissues induced by higher auxin concentration, which ceases the regeneration ability of tissues (Baker and Wetzstein, 2004). Our results are in agreement with those reported by Grigoriadou et al. (2011), where the addition of 5 μM BA + 2.5 μM IBA (cytokinin/ auxin, 2:1 ratio) to the modified MS medium (x 2Fe) was the most appropriate proliferation treatment for A. occulta (3.5 new shoots/ explant 0.93 cm long). The in vitro culture of A. biebersteinii was best promoted by adding 8.8 μM BA to the MS basal medium (Ghasempour et al., 2012).

Different agar concentrations
In this study, the percentage of explants with multiple shoot formation and the length of A. occulta microshoots was adversely affected when cultured under the highest agar concentration of 8 g L -1 . There is a reverse analogous relationship between the gelling agent concentration and water potential -water and dissolved substances availability, thus the increase of the former results in the decrease of the latter (Smith and Spomer, 1995). The proliferation rate of A. occulta microshoots under study was substantially raised under the intermediate agar concentration of 7 g L -1 , however the further increase in its concentration to 8 g L -1 had a negative effect. Shoot proliferation rate of plantlets cultured in vitro is reported to be declined with the increase in agar concentration (Yadav et al., 2003) and the reduced regeneration capacity of explants could be ascribed to the lowering of water potential in the medium, derived from the increase in the osmotic potential imposed by higher gelling agent concentrations than the optimum (Smith and Spomer, 1995).

Different cytokinin types
In this study, BA at 4.4 μM gave better multiplication results as evidenced by the higher shoot number and proliferation rate than 4.4 μM KIN when combined with 0.44 μM IBA. Similar observations that are in line with our findings have been found by Marcu et al. (2014), who reported that BA was more favorable for shoot proliferation of in vitro cultures than KIN, and such response could be explained according to Buah et al. (2010) by the higher stability and thus longer persistence of BA in the medium. However, different cytokinin types, other than BA, were shown to be more beneficial for shoot proliferation attributes of in vitro cultures in other Achillea species including 5.6 μM KIN in A. fragrantissima (Younes et al., 2015) and 3.4 μM TDZ in A. millefolium L. (Alvarenga et al., 2015). The optimum concentration of PGRs for inducing shoot proliferation is species-dependent since KIN concentration was demonstrated to be higher than BA for exhibiting the same optimum result regarding multiple shoot formation and elongation in Portulaca grandiflora (Srivastava and Joshi, 2009). All four different cytokinin types studied herein had a positive effect as exhibited higher shoot numbers and proliferation rates in comparison to the control (cytokinins-free) with BA to be the most effective, whereas non-significant were the changes related to shoot length. The best conditions for in vitro shoot proliferation in other Achillea species were 4.4 μM BA + 2.8 μM IAA for A. filipendulina (Evenor and Reuveni, 2004), and 4 μM BA for A. millefolium, thus BA proved to be useful for micropropagation in comparison to either ZT or KIN (Shatnawi, 2013). In the studied A. occulta, shoot number was better stimulated by BA (2.55) compared with KIN (1.85), the length of produced shoots was higher in the presence of ZT (0.70 cm) than KIN (0.59 cm), proliferation rate was the highest with BA (1.66), while non-significant changes were noticed in shoot formation percentages (60-85%) irrespective of cytokinin type (BA, 2-ip, ZT, KIN). Based on the results obtained by Turker et al. (2009) in A. millefolium L., shoot-tip explants when cultured on MSMO medium enriched with either 13.32 μM BA + 2.85 μM IAA or 23.23 μM KIN + 4.92 μM IBA gave the highest shoot numbers at a 100% percentage. Zebarjadi et al. (2011) pointed out that the percentage of multiple shoot formation may be influenced only when the endogenous cytokinin level in the plant is low, therefore if this condition is not valid and its content inside the plant is high then the exogenous application of this plant growth regulator in the culture medium may have negative impact on proliferation. The differential response of the explants to different cytokinin types may be explained by differences occurring in their uptake, recognition by the cells, and/or the mechanism of the cytokinins action (Kim et al., 2001). BA was the most effective cytokinin type for proliferation of A. occulta under study, followed by ZT, while KIN was the least effective. The superiority of BA, over the other cytokinins, might be attributed to its ability to increase the polyribosome content in the cells due to the production of D-type cyclin (CycD3), which stimulates the cell cycle progression (Pasternak et al., 2000).
ZT application method and BA concentration Filtration through microporous filters (0.22-0.45) is used for thermo-labile organic constituents such as vitamins, growth regulators and amino acids (Torres, 1989). Physical stability of adenine-based cytokinins (BA, KIN, 2-ip, ZT, TDZ) as affected by high temperatures and pressures, such as those within an autoclave cycle is of interest (Hart et al., 2016). Even though the stability of KIN (Miller et al., 1955) and zeatin riboside (Miller, 1974) during autoclaving has been studied, the majority of cited literature is anecdotal whether these cytokinins are heat-labile i.e. ZT and 2-ip (Iliev et al., 2010) or not heat-labile i.e. BA and KIN (Panaia et al., 2011). In the present study, shoot formation percentages and shoot numbers per explant were not affected from either ZT sterilization method (autoclaved, filtered) or cytokinins application form (BA, TZ, BA + TDZ). In consistency with our findings, non-significant was the difference in the results obtained in a soybeanbioassay in the case of sterilization method of zeatin riboside, exogenously applied either through autoclaving or filtration, without however measuring quantitatively or specifically its degradation (Miller, 1974).
In the studied A. occulta, on the one hand, shoot length was better stimulated when ZT was autoclavesterilized and applied as a single cytokinin type, while filter-sterilized ZT without undergo autoclaving (1.1 bar, 121 °C for 20 min) adversely affected this parameter. In the same study, on the other hand, different response on shoot length was observed in the combined effect of both cytokinin types, BA + ZT, regardless of ZT sterilization method being non-statistically significant. According to Hart et al. (2016), the stability of ZT in MS medium-basal salts after one autoclave cycle of 1.1 bar 121 °C for 30 min revealed negligible amounts of degradation when dissolved at 10 mg L −1 KOH. In our study, ZT before its incorporation into the culture medium, either autoclave-sterilized or filter-sterilized, it was dissolved in a relatively high base concentration (1.0 N KOH). The concentration of solvent in the case of KOH base for dissolving ZT prior its sterilization plays a significant role in the rate of its degradation loss, thus 20% of its mass can be lost over 18 days after being dissolved in 0.5 N KOH (Hart et al., 2016).
Although ZT sterilization method hardly affected proliferation rate of the studied A. occulta, autoclavesterilized ZT vs filter-sterilized ZT, and autoclave-sterilized ZT + BA vs filter-sterilized ZT + BA, the factor that played the key role was the presence of a single cytokinin (BA or ZT) or combined cytokinins (BA + ZT), regardless of ZT application method. It becomes clear that the stability of adenine-based cytokinins such as ZT in a range of aqueous solution conditions and through one autoclave cycle in a moderately alkaline solution (0.05 N KOH) is depended on several factors including crystallization and acidic conditions during extreme heating, which can lead to potential adenine degradation, therefore these compounds should be used with caution and administered properly for plant tissue culture (Hart et al., 2016). In several woody species and in accordance with our findings, the combined use of ZT with BA proved to be more efficacious for shoot proliferation than ZT alone (Grigoriadou et al., 2002;Ali et al., 2009).
The effectiveness of various combinations of cytokinins on micropropagation of woody plants has been reported by Higuchi et al. (2004), however, shoot elongation phase is highly sensitive to higher concentrations of growth regulators (Kadota and Niimi, 2003). In this study, autoclave-sterilized ZT enhanced better the elongation of produced A. occulta microshoots, while BA applied either alone or with ZT (autoclave-or filtersterilized) had a negative effect. A possible explanation for this outcome is that the excessive supply of both cytokinins (ZT + BA) has concealed the balance of cytokinins resulting in ethylene production which in turn inferred the inhibition of internode elongation due to cessation of cell division and expansion, suggesting interdependence of hormonal regulatory pathways (Higuchi et al., 2004). Our results are not in line with those reported by Rugini et al. (1999) and Ali et al. (2009), as shoot elongation response to the combined use of ZT + BA was more effective than that of ZT alone, showing effect species-dependent.

In vitro rooting under different auxin types and concentrations
The exogenous application of auxins is essential especially in the early stages of rhizogenenis (Radmann et al., 2014) and the emergence of numerous roots (Dobranszki and Silva, 2010) including IBA and NAA as synthetic auxin types, and indole-3-acetic acid (IAA) as the main auxin type occurring naturally within plants (Phillips and Garda, 2019). Regarding the studied A. occulta, IBA was the ideal auxin type and 2 μM its optimum concentration for maximum response related to rooting percentage (20%). Our findings are in line with the outcomes derived from a previous study conducted in the same plant species (A. occulta), wherein rooting of microshoots proved to be very difficult exhibiting a 12.5% rooting percentage after supplementing the MS medium with 20 μM IBA (Grigoriadou et al., 2011). There are several reports in other Achillea species pointing out that the highest rooting percentages were achieved with 9.84 μM IBA in A. biebersteinii (Kahrizi and Kakaei, 2010), IAA (2.85 or 5.72 μM) or 2.69 μM NAA in A. filipendulina (Evenor and Reuveni, 2004), 9.84 μM IBA (Turker et al., 2009;Shatnawi, 2013) and 3.23-10.74 μM NAA in A. millefolium (Shatnawi, 2013), and 5.72 μM IAA in A. pyrenaica Sibth. ex Godr (Marcu et al., 2014). The better effectiveness of IBA compared with NAA on rooting induction of A. occulta in this study could be attributed to several reasons including (1) more resistance to photodegradation, adherence to microshoots, and inactivation by biological action (Elmongi et al., 2018), (2) successive rooting gene activation, better uptake, transportation and stability (Liu et al., 2017), (3) slow migration and delayed degradation (Mansseri-Lamrioui et al., 2011), and (4) slower oxidization rate by the oxidase (Wolella, 2017) and thus of higher stability (Abbasi et al., 2019). It this study, the highest concentration (5 μM) of either IBA or NAA, and the simultaneous application of both auxins (IBA + NAA) irrespective their concentration combination ratios were detrimental and toxic resulting in complete rooting inhibition, which might be ascribed to ethylene synthesis induced due to high auxin concentrations (Aghaye and Yadollahi, 2012).
Root elongation of A. occulta microshoots under study was best stimulated with 2 μM IBA. It has been formulated that low auxin concentrations are promotive on root growth and inhibitory at high ones (Baker and Wetzstein, 2004). Similarly to ours results, 9.84 IBA had the best effect on root length in A. biebersteinii, whereas higher concentrations had a negative effect (Kahrizi and Kakaei, 2010). Different auxin types, other than IBA, were found to be more effective on root elongation in other Achillea species including IAA in A. filipendulina followed by IBA as compared with NAA (Evenor and Reuveni, 2004) and NAA in A. millefolium (Shatnawi, 2013). The increase of NAA concentration was accompanied by a successive decrease of root length in this study and this might be due to differences in the catabolism of this auxin (Evenor and Reuveni, 2004) as a result of differential transport of the various auxins (Yamamoto and Yamamoto, 1998).
In the current study, NAA was the ideal auxin type and 2 μM its optimum concentration for maximum response related to root number as compared to IBA regardless concentration. Our results come in contradiction with the results reported in other Achillea species including A. filipendulina where all three auxin types used (IBA, NAA, IAA) gave similar root numbers (Evenor and Reuveni, 2004), A. millefolium where root number was maximum with 6.85 μM IBA (Shatnawi, 2013), and A. pyrenaica where 5.7 μM IAA gave more roots compared with 5.4 μM NAA (Marcu et al., 2014).

Conclusions Conclusions Conclusions Conclusions
In this study, a successful reproducible micropropagation protocol of A. occulta is presented enabling its large-scale multiplication, domestication and germplasm conservation. The direct organogenesis protocol was established based on several culture media compositions and parameters for optimization, including different types and concentrations of PGRs as well as their ratio, different agar concentrations, different cytokinin types, different sterilization methods of ZT prior its addition to the culture medium, as well as different auxin types and concentrations. The optimized micropropagation protocol established in this study could be used as a first step tool in the future for the large-scale production of certain secondary metabolites from in vitro grown A. occulta plants contributing to further analyses related to their pharmacological, physiological and biochemical background with potential use in dermatology and cosmetic industry. Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable.