Anise-Pimpinella anisum L. (Umbelliferae, Apiaceae) is an important flowering, medicinal and aromatic plant native to the Eastern Mediterranean and South Western Asian regions [1,2] that grows to around 40–50 cm with white or yellow flowers [3]. It has been cultivated in Egypt for the last 4,000 years; from where it spread to other parts of Europe and the Middle East [4]. The species belonging to the genus Pimpinella are represented by 27 taxa in the flora of Türkiye [5] and have multiple uses in ethnomedicinal systems. It is used singly or in combination with other herbs and taken as a carminative stomachic, stimulant, expectorant, antispasmodic, and antiseptic [6]. It is also used as an aromatic flavoring agent in desserts and alcoholic beverages [7]. The plants grow and establish well on fertile, warm soils soon after spring [8].

Anise seeds have very small endosperm, and face difficulties during early stages of growth. Therefore seeds face multiple abiotic and biotic stresses; which may inhibit their germination, along with growth and development of seedlings under natural conditions [9,10]. Several strategies in soil fertilization are being employed by farmers to improve germination and emergence that vary depending on the farmers. Most of the farmers always tend to apply an easy and cheap way of crop production. Seed pre-sowing osmopriming treatments are widely used as an easy way to biofortify seeds with nutrient elements to stimulate their germination, emergence [11] and inhibit the effects of external stresses [12].

Hydroprimed and osmoprimed seeds of Oryza sativa L. [13,14], Brassica juncea L. [15], and Lens culinaris Medik. [16,17] showed improvement in germination and stand establishment. Although, anise is used as a multifunctional medicinal crop like Nigella sativa [18] and Ocimum basilicum [19] the studies on germination, seedling establishment and agronomic practices are rare. There is no study regarding the strategy of treating MgCl₂ (Magnesium chloride), CaCl₂ (Calcium chloride), and ZnCl₂ (Zinc chloride) to improve anise germination and protect them from stresses during germination. This study evaluated the metabolic stimuli induced changes in anise seeds due to MgCl₂, CaCl₂, and ZnCl₂ based osmopriming for obtaining uniform seed germination and stands in fields that are difficult to obtain under natural conditions [20]. These stored mineral nutrients like magnesium, calcium, and zinc have importance especially when seedlings are under conditions of limited nutrients. Enrichment of seeds via seed priming with these nutrients could promote germination and seedling growth.

Magnesium (Mg) is an important macronutrient element that is required to carry out several biochemical and physiological processes in plants [21] during photosynthesis by binding to chloroplasts, participating in the light harvest in PSI and PSII [22]. It is also involved in carbon fixation by chloroplasts [23]. Mg is a micronutrient element and an environment-friendly biostimulant. Mg deficiency reduces the rate of the biomass with a disruption of CO₂ fixation and generation of reactive oxygen species (ROS) ending up in cell damage [22]. It also acts as an enzyme cofactor activity with ATP [24] based on hydrolysis and synthesis [25]. It is an important constituent of chlorophyll molecules and the powerhouse behind photosynthesis.

Calcium (Ca) is one of the main components of cell structure and it takes a role in cell elongation, division, controlling nutrient absorption, and helping water absorption [26]. Furthermore, due to its role in cell structure, and signaling roles in plants; Ca²⁺ ions are important in charge, osmotic balance, and in seed germination. When the seeds are imbibed with the impact of rehydration, gibberellins cause Ca²⁺ flux into the cytosol and the expression of calmodulin (Ca²⁺ binding proteins); which are involved in signal transduction [27,28]. Alpha-amylase enzyme one of the Ca²⁺ metalloenzymes required at the beginning of germination [29,30], is the consequence of Ca²⁺ related post-imbibition metabolism.

Zinc (Zn) is crucial during seed germination and the early growth stage of plants until the root system gains the ability to nutrient uptake from the soil [31]. Zinc is a co-factor of various enzymes that take a role in the detoxification of ROS. Beyond that stage, the seeds containing low zinc show a delay in germination and poor seedling vigor [31,32].

Chlorides (Cl) prevent the accumulation of free amino acids and protect plants against diseases with easy management of intercellular transport of water [33]. It is understood that both Mg and Cl ions could improve quality and yields in cereals and other crops.

Therefore, the present study aimed to evaluate the impact of water, MgCl₂, CaCl₂, and ZnCl₂ priming on anise seeds germination and seedling growth under controlled conditions.

Seed water uptake varied among treatments (p = 0.0000). Seeds soaked in water for 8 h had the highest water uptake (96.00%) while the minimum water uptake (56.24%) was observed in 4 h CaCl₂ soaking (Table 1).

Mean emergence time represented a statistical difference over seed treatments (p < 0.0000). A comparison among seed priming treatments showed statistical differences among the non-treated, hydroprimed, and osmoprimed seeds. Non-primed seeds took 11.20 days to emerge. 8 h MgCl₂ priming also shortened the mean emergence time over control, which was statistically different from non-treated control treatment and were statistically different among themselves showing a range of 9.73 to 17.09 days to emerge. The minimum and the maximum time to emergence were noted in 8 and 2 h MgCl₂ priming. CaCl₂ and ZnCl₂ priming did not show any superiority over control or 8 h MgCl₂ priming and the emergence time was slower (Table 2).

Impact of seed treatment on the emergence percentage showed statistically significant differences (p < 0.0000). The control and MgCl₂ treatments showed maximum and statistically similar values of 98.00% and 97.50%, respectively. The hydroprimed seeds were negatively affected in terms of the emergence percentage compared to the non-treated seeds (control treatment). The minimum emergence percentage of 48.50% was noted on 8 h of hydroprimed seeds. Emergence percentages of CaCl₂ primed seeds varied between 54.00% to 68.50% and ZnCl₂ primed seeds varied between 54.50% to 67.50% (Table 2).

The root length of anise seedlings showed significant differences (p < 0.0000) among non-treated, hydroprimed, and osmoprimed seeds (Table 3). A comparison of the mean root length values showed that the maximum (12.90 cm) root length was noted from 8 h of MgCl₂ primed seeds. Minimum root length was noted (5.43 cm) on 8 h ZnCl₂ primed seeds. Hydroprimed and CaCl₂ primed seeds had the longest roots in 8 and 2 h duration in the same order.

Analysis of variance showed significant differences (p < 0.0000) among treatments for shoot length. The maximum shoot length value of 10.77 cm was observed in the control (non-treated) treatment. This value was statistically similar to the 2 h hydroprimed seeds with values of 9.59 cm, 4 and 8 h MgCl₂ primed seeds with values of 10.18, 10.35 cm and 2 h CaCl₂ and ZnCl₂ primed seeds with values of 9.62 and 10.43 cm. The minimum shoot length value of 6.92 cm was noted from 8 h hydroprimed seeds. It seemed that the negative effects of hydropriming on growth parameters were due to over-imbibition by anise seeds during hydropriming (Table 3).

Seedling fresh weight was significantly (p < 0.0000) affected by seed treatments (Table 3). The maximum seedling fresh weight value of 256.30 mg plant⁻¹ was noted in 8 h MgCl₂ treated seeds, which showed statistical similarity with non-treated seeds (254.00 mg plant⁻¹), 2 h hydroprimed seeds (237.50 mg plant⁻¹), and 2 h ZnCl₂ treatment (253.30 mg plant⁻¹). The lowest seedling fresh weights were obtained from 8 h ZnCl₂ primed seeds with a value of 112.30 mg plant⁻¹.

The seedling dry weight values of anise were significantly affected (p < 0.0000) by seed treatments. While the maximum seedling dry weight value was observed in non-treated seeds (26.25 mg plant⁻¹), statistical similarity with 8 h hydropriming (24.00 mg plant⁻¹) and 8 h MgCl₂ (23.00 mg plant⁻¹) treatment were also observed (Table 3).

Results about chlorophyll contents measurements (Table 4) clearly reflected the sharp impact (p < 0.0000) of seed treatments on chlorophyll contents. The maximum chlorophyll contents in terms of SPAD unit were observed in 4 h CaCl₂ priming with 35.69 SPAD units and statistically similar values were noted in control (34.51 SPAD units) and 8 h MgCl₂ priming (35.18 SPAD units). The osmoprimed seeds improved the chlorophyll contents depending on the duration of treatment.

The electrical conductivity test showed significantly dissimilar variations (p < 0.0000). The minimum values were observed in MgCl₂ priming in all treatment durations between 9.71–9.99 μS cm⁻¹ (Table 4). The maximum EC value was observed in 8 h hydropriming with 671.7 μS cm⁻¹.

During priming treatments, the seeds were partially hydrated with water and different osmotic agents to a point (2, 4, and 8 h). Water uptake showed diversity among treatments. The results revealed that among priming treatments hydropriming had the highest water uptake in all durations. This could be attributed to a low EC value of distilled water compared to other treatments, leading to uncontrolled water uptake.

MgCl₂ priming treatments showed an increase in root length, fresh and dry weight along with chlorophyll contents with each increase in priming treatment duration of 2 to 8 h. CaCl₂ priming treatment showed maximum improvement after 4 h of priming treatment. These two treatments were at par with the values obtained from nonprimed seeds used as control treatment. ZnCl₂ priming treatments were noneffective at any duration of treatment and were inferior compared to the control treatment. The maximum values for these parameters were noted on 2 h treatment duration followed by obtaining decreased values for each parameter on 4 and 8 h priming duration treatments. The results indicated variable inhibition in these parameters for the rest of the primings for 2, 4, and 8 h excluding 8 h MgCl₂ treatment.

Seedling dry weight in control had the maximum value among all treatments but no statistical difference was observed with 8 h MgCl₂ priming. Priming duration in each treatment showed dissimilar results. This showed that the seeds could end up with either of the

replacement of seed nutrient matters in the cytoplasm with water moving from outside.

An imbalance in the solid loss/water gain ratio is not desired and ends up in embryo damage with negative implications on the growth of seedlings’ roots and shoots depending on the environmental factors and genetic potential of the seeds [39,40].

Mg has an important role in the development and formation of sink organs [41,42] and the extra Mg given through osmopriming helped seed and seedling attributes to improve them in terms of fresh seedling weight per plant over other treatments. The nutrient matters are lost and washed away through osmosis with each increase in the duration of hydropriming, which influence the growth and development of roots from the seeds [42]. MgCl₂ osmopriming maintains the genetic fidelity of the seeds, which should be carried out very carefully. Taking water up to a critical point is essential for germination and improving seed vigor, with better root growth and uniform crop establishment, and larger canopy [43,27]. This helps in improved competition with the surrounding plants and higher yields [44].

Zinc is important during germination against abiotic stress and for seedling vigor. However, ZnCl₂ priming induced inhibition, toxicity, and dormancy during germination and growth compared to the control or other priming treatments. It seemed the amount and duration of ZnCl₂ (100 mM) treatments used in the current experiment were toxic for anise. These findings were corroborated by Stanković et al. [45] who had similar findings using different levels of ZnCl₂ to germinate wheat seeds germination observing limited seedling growth.

Hydropriming during 2, 4 and 8 h led to uncontrolled water uptake of anise seeds and less emergence percentage than other treatments. Imbibition damage was also evident due to minimum germination in 8 h hydropriming. Imbibition damage due to uncontrolled rapid water uptake causes cell death and high solute leakage which was observed as the highest in hydropriming compared to other treatments. Considering these results 8 h of priming with water reduced the emergence percentage for anise seeds. The disadvantages of hydropriming were mentioned by Lutts et al. [46] and it is also recommended to define accurate treatment duration, temperature, and water volume for every species. On the contrary osmopriming with 8 h MgCl₂ created a positive impact on emergence percentage due to slow water uptake related to low water potential. Positive effects of MgCl₂ seed priming on germination percentage have been noted in rice by Brooks et al. [47].

A decrease in chlorophyll content leads to low photosynthesis which has a negative impact on plant growth [37]. Magnesium is necessary for the synthesis of chlorophyll, and essential for photosynthesis [14] and its deficiency causes a reduction in chlorophyll concentrations in leaves [42]. Additionally, Wang et al. [48] have already mentioned the protective role of Ca²⁺ on photosynthetic electron transport. The results show similarity with the observation showing 8 h MgCl₂ and 4 h CaCl₂ priming of anise seeds led to an increase in chlorophyll synthesis compared to hydroprimed seeds in this research.

Electrolyte leakage has been reported as the determiner of seed viability and vigor in several species. The seeds having less vigor leak more electrolytes during imbibition [49,50]. The hydroprimed anise seeds had less emergence percentage and had the maximum EC values which seemed as an indicator of hydropriming with a negative impact on anise seeds and germination. However, priming with MgCl₂ gave the minimum EC values among priming treatments, which was the evidence of repair mechanism of the membrane, especially for 8 h MgCl₂ priming.

Sustaining favorable water under field conditions is critical for the germination process. Priming with different osmotic agents and water generated moderate abiotic stress (osmotic stress, saline, and drought stress) during soaking, and accumulation of osmotically active solutes like proline, which is reported in a dissimilar number of species during seed priming [51–53,46,42].

Any duration of hydropriming treatments is not recommended for anise. It seemed all treatment durations in hydropriming had inhibitory effects due to imbibition-based damages. This trend was also indicative in all osmoprimed seeds, excluding 8 h MgCl₂ priming treated seeds. Although 8 h MgCl₂ priming treatment and non-treated control treatment were statistically similar the former showed a numerical improvement over the other. This showed that biofortification of MgCl₂ improved anise seed germination and emergence.