Secondary metabolite changes in Maymars juniper cuttings ( Juniperus sabina ) under diﬀerent treatments of propagation (IBA, substrate and har­ vest time of cutting)

: The endemic Juniper of Maymars ( Juniperus sabina ) is one of the most valuable plants in forested areas. The objectives of this experiment were: I) to determine the best conditions for stem cutting propagation of this species, and II) to examine changes in some of the secondary metabolites during the four months (the ﬁrst of each season): January, April, July, and October, after rooting of cuttings. The research was done with the treatment of ﬁve levels of Indole Butyric Acid, including: 0, 1000, 2000, 4000, and 8000 ppm in four root­ ing substrates, including perlite, perlite­cocopeat (1:1), pumice, and a mixed rooting substrate (sand, perlite, cocopeat, vermicompost, and potash; 1:1:1:1:1) in the four seasons of the year, with stem cuttings having an average length of 15 cm. The best treatment with more than 50% rooting was seen in April at levels of 4000 and 1000 ppm, and the best substrate was perlite coco­ peat. Using lower levels of IBA led to a reduction in total phenol


Introduction
The genus Juniperus is one of the few conifers that act as a main tree in the natural ecosystems of the mountainous forests of the world. The protective and valuable roles of various species of junipers in the management of forest erosion and water management are well known. Also, the role of junipers is important both in water storage and in soil conservation (Ali Ahmad Koruri et al., 2011). They are great landscaping and ground cover species (Westerfield, 2012). Among the junipers, Juniperus sabinaMaymars is one of the most popular types of junipers. This species can be utilized for forest restoration on poor sites with low potential produc tivity, such as arid and semiarid areas. In addition, Maymars is one of the most beautiful juniper species and is suitable for ornamental use (Piotto and Di Noi, 2003). Thus, information about the plant production of Juniperus sabina can be useful for forest managers and plant producers in many areas.
Berry extract of Juniperus sabina showed inhibito ry activities against KB tumor cell lines (Sadeghiali abadi et al., 2009). Fruit and leaves of junipers are commonly used as tea and pounded fruits are eaten to lower blood glucose levels in Anatolia. To evaluate antidiabetic and antioxidant potential and the chemi cal profile of Juniperus sabina L. in a study, phyto chemical screening tests indicated the presence of flavonoids, tannins, terpenoids and carbohydrates in the extracts (Orhan et al., 2017).
Maymars juniper is usually propagated by vegeta tive methods (Gheorghe et al., 2010). To propagate plants via cuttings, the indolebutyric acid (IBA) growth regulator has been used as a treatment (Amri et al., 2010). To produce junipers by stem cuttings, IBA has been used in previous studies (Henry et al., 1992;Rifaki et al., 2002). Research conducted by Rifaki et al. in 2002 on vegetative propagation showed the best concentration for the cuttings of junipers at 4000 ppm of IBA.
Phenolic compounds have effects on growth, development, propagation and plant defense (Croteau et al., 2000). Measurement of internal com pounds and their comparison during growth or root ing can be valuable factors in identifying internal bar riers or enhancers of rooting in the cuttings, as there are no extensive resources available in this regard.
Phenolic compounds are a group of antioxidant agents (Choudhury et al., 2013). Many scientists have reported the relationship between total phenol and antioxidant activities (Hariprasath et al., 2015). In the propagation of varieties of blueberry, softwood cut tings and tissue culture, the interaction of genotype, propagation methods, and growth seasons signifi cantly affected flavonoid content and antioxidant capacity. The interaction effect of the propagation method and genotype significantly affected total phenol and chlorophyll content. Also, the interaction between propagation method and growth season sig nificantly affected the total flavonoid content (Goyali et al., 2013).
Some studies have also revealed differences in rooting of cuttings as affected by substrate (Kentelky, 2011). Cocopeat and IBA were used to propagate Juniperus excelsa through stem cuttings, and they improved rooting ability (Esmael Nia et al., 2006). Growth regulator and substrate are effective on the rooting of the cuttings of Juniperus oblonga, and proper substrate composition and the use of benzyl adenine increase the rooting of the cuttings (Khoshnevis et al., 2012).
Roots uptake minerals and water from the soil (Chapin et al., 1987). Higher numbers of adventitious roots could improve the root system's symmetry, sta bility, survival, and growth rate (Bryant and Trueman, 2015). Thus, rooting percentage is a good indicator of the growth strategies of root development and the capacity to endure water stress in Juniperus trees (Garcia Morote et al., 2012). Therefore, the present study is intended to inves tigate an efficient method of vegetative propagation of Maymars juniper using stem cutting and its effects on some of its phytochemical characteristics (pheno lic compounds). We hypothesized that high level of phenolic compounds during rooting can be an indica tor of the level of rooting in cuttings of Juniperus sabina, and that the percentage of rooting should be an indicator of rooting performance in cuttings. Thus, the objective of this research was to analyze the effects of five concentrations of IBA as treatment and four substrate types (perlite, perlite cocopeat, pumice, and mixed substrate) on the level of pheno lic compounds and rooting performance in cuttings. The experiment was conducted in four months (February, mild climate; July, warm temperate cli mate; October, relatively cold weather; and January, cold weather) to determine the impact of harvesting time on the rooting capacity of cuttings.

Materials and Methods
Cutting preparation, treatment with indole butyric acid (IBA), and substrate composition.
The cuttings of Juniperus sabina were sampled from its natural habitat in the Chaharbagh mountains of Gorgan, North Iran ( Fig. 1), one of the main Mediterranean populations at higher altitude (2,700 m a.s.l.). Using a 30year average, the mean annual temperature at the site is 9.2°C, and the mean annu al precipitation is 429 mm. Extreme temperatures (summer and winter) range from 23°C to 5°C (data from Gorgan climatic station: 46° 06 N, 28° 00 W; 2,600 m a.s.l.). The crowns are approximately 2 x 2 m in length and width. The ring diameter of shrubs is 20.0 cm averagely, and the height is 1.5 m (these are old and horizontal shrubs). Generally, 20 male shrubs have been used for this experiment, and they are all growing in the same area with the same ecological environment. The experiment was conducted at Gorgan University of Agricultural Sciences and Natural Resources in winter, spring, summer, and fall of 2017. Stem cuttings were only collected from the upper crowns of male trees.
Cuttings were harvested in the morning. After har vesting, the stem cuttings were prepared to be 15 cm in length and 0.50.7 cm in diameter (Bohlenius et al., 2017) for treating and cultivation in a greenhouse. Substrates were prepared, and cuttings were placed in the greenhouse equipped with an automatic sys tem to control humidity (micro irrigation) and bottom heat. The average daily temperature during the experiment was 22°C, and the average relative humidity was 77%. The amount of light entering the greenhouse was varied based on the amount of nat ural light in each season.
For the treatment of stem cuttings, five levels of IBA were used: 0 or control, 1000, 2000, 4000, and 8000 mg L 1 (Control is a sample that is placed in the substrate without adding any treatment and is used to compare the effect of the treatments used on cut tings). The base of each cutting was placed in the aqueous solution of IBA for five seconds and then inserted into the substrate. The four used substrates were: I) perlite; II) mixed rooting substrate a combi nation of sand (20%), perlite (20%), cocopeat (20%), vermicompost (20%), and potash (20%); III) perlite cocopeat (1:1), and IV) mineral pumice (each sub strate about 10 Kg). For each treatment (combination of treatment and substrate), three replicates were prepared, with nine cuttings per replicate. Thus, a total of 540 cuttings in each season were cultivated.

Total phenol, flavonoids, and antioxidants of stem cuttings
Secondary metabolites were measured in both rooted and unrooted stem cuttings to detect differ ences in the internal compounds between cuttings that have the potential for rooting and others with out this potential. For evaluating the treatments and to make comparisons between the chemical com pounds in cuttings at the beginning of the sampling and the amount of increase or decrease between the time of planting and rooting (between the first and the end of each season), samples were taken from freshly harvested cuttings in each season (the first of each season with samples separately from the stem cuttings) and compared with the results at the end of the growing season.
In order to measure total phenol, antioxidants and flavonoids (at the end of each season and after harvesting the cuttings from substrate), in the first step, one gram of each plant sample, which was the bark of the stem of each cutting separately, was removed and powdered with liquid nitrogen, then placed in 10 cc of 80% methanol (Merk) in an Erlenmeyer flask, and after that, placed on a shaker for 24 h. The mixture was then filtered with filter paper and clean extracts were used to measure sec ondary metabolites in mg/g fresh weight (McDonald et al., 2001). Then we began to assess the total phe nolics, antioxidants, and flavonoids.
To measure total phenol, 20 μl of each of the above plant extracts were added to 1.16 μl of dis tilled water, 100 μl of folin (Merk) and 300 μl of sodi um carbonate (20%), and they were mixed in a test tube (it is done for each plant sample separately) and then placed in a water bath at 45 °C for 30 min. After that, each sample was measured by a spectropho tometer (UnicUV 2800 4 cells) at a wavelength of 760 nm. After drawing the standard graph (prepara tion of different concentrations with specific values of the controldifferent samples and readings with the spectrophotometer and then drawing on the curve) (Fig. 2  To measure the flavonoids, 0.5 ml of each plant extract, 1.5 mg/L pure methanol (Merk), 0.1 ml of aluminum chloride, 0.1 ml of potassium acetate, and 2.8 ml of distilled water were combined and mixed in a test tube, and then all samples were placed in the dark for 30 minutes, and after that, they were mea sured by a spectrophotometer with a wavelength of 415 nm. After drawing the standard graph (prepara tion of different concentrations with specific values of the controldifferent samples with readings with the spectrophotometer and then drawing on the curve), the flavonoid value of each sample was obtained (Chong et al., 2002) (Fig. 3).
To measure antioxidant activity, 1 ml of each plant extract was removed. In the next step, the amount of 0.0004 mg of DPPH was dissolved in 10 ml of methanol (Merk), and then 1 ml of this solution with 1 ml of each extract of the plant previously removed was combined, and finally, the antioxidant percentage was measured in a spectrophotometer with a wavelength of 517 nm (Miliauskas et al., 2004).

Rooting percentage
To determine the rooting percentage of each treatment, the roots were counted in all rooted cut tings ( Fig. 4) in each treatment (three replications and each replication contained 9 cuttings; totally 27 cuttings) and then this number of cuttings was divid ed by 27 (some cuttings were unrooted and some of them were dried), (Negash, 2002).

Statistical analysis
A factorial arrangement of treatments (Hoshmand, 2006) was applied to analyze the effects of three main factors on five dependent variables. The first factor was "treatment" or concentration of IBA (five levels: 0, 1000, 2000, 4000, and 8000 ppm), the second was "substrate" (four levels: perlite, per litecocopeat, pumice, and mixed rooting substrate), and the third factor was "season" (four levels: January, April, July, and October). This represents a 5 x 4 x 4 factorial with 80 combinations of factor levels or treatments. The dependent variables were inter nal compounds of the cuttings (secondary metabo lites in both unrooted and rooted cuttings) and the indicator of rooting performance (% of rooting). Therefore, in the dependent variables concerning chemical internal compounds, another level was added as treatment, secondary metabolites in fresh samples (stem cuttings not planted and prepared at the beginning of each season). This was done to com pare the effects of treatments between cuttings not treated (at the beginning of each season) and treated cuttings at the end of each season.
SAS® statistical software (Neter et al., 1996) was used to detect significant factors and to compare mean values between factors and levels of treat ments. The comparison of the means was done using the PROC GLM procedure. We utilized Multifactor Analysis of Variance (a threeway ANOVA model) at a probability level of 5% (p<0.05). The analysis within season was performed by a twoway ANOVA (exclud ing season as a main factor in the complete model).
In this research, we performed independent ANOVAs (not a mixeddesign nor a repeatedmeasures ANOVA) because the measurements were indepen  dent (we used different stem cuttings for each treat ment and season).
A Fisher's Least Significant Difference (LSD) test (p<0.05) was used to determine the significant differ ences between treatments (Neter et al., 1996). To apply this statistical method, it is desirable for data to be normally distributed. This is not the case with pro portions, which have values that range between zero and one. In addition, errors must be independent and normally distributed with constant variance. To ensure these assumptions, a logarithmic transforma tion was used (Sabin and Stafford, 1990): for the per centage of rooting, the analyzed variable was [ln (r+0.5)], and r was the percentage of rooting (divided by 100). As this transformation requires numerical data above zero, a small number (0.5) was added to this variable before the transformation. The other dependent variables were normal and then distrib uted.

Rooting performance
In Table 1, the pvalues for the three principal effects (substrate, treatment with IBA and season, and their twoway interactions) and for the effects within each season (substrate, treatment, and their interactions) are represented. Effects must be consid ered significant when p<0.05. 540 stem cuttings in each season were planted. In January, five treat ments rooted (99 cuttings), and 441 cuttings were unrooted. In April, 20 treatments rooted (502 cut tings) and 38 cuttings were unrooted. In July, four treatments rooted (89 cuttings) and 451 cuttings were unrooted. In October, four treatments rooted (102 cuttings) and 438 cuttings were unrooted.
As it is clear from figure 5, the best rootgrowing month is April. During spring, rooting was more than 50% at a level of 4000 ppm of indole butyric acid with no significant difference at the 1000 ppm level. Also, the minimum rooting percentage of the cuttings in this month was about 25% at the level of 8000 ppm of indole butyric acid and control treatment; howev er, it was higher than the rooting percentage of other months. In the study of the effect of different sub strates on the percentage of rooting of the cuttings, the best substrate was seen in equal parts of perlite cocopeat (v/v), with rooting at a maximum of 62% with a treatment of 1000 ppm (Fig. 6). And this sub strate was one of the substrates that had the largest number and length of roots (Fig. 7 C and Fig. 8 C). Therefore, among the substrates used in this research to root the Juniperus sabina, the best sub strate was perlitecocopeat, with a maximum rooting percentage of 98. While the least rooting percentage of cuttings was seen in January, with less than 2% in all treatments, October is also not a good time for the reproduction of this plant. On the other hand, the most root number and root length was seen in April ( Fig. 7 B and Fig. 8 B). So, the best months for rooting of cuttings of Juniperus sabina are April and May, and the best levels of IBA used were 4000 and 1000 ppm, Despite the fact that the largest number of roots was not seen in these treatments.

Secondary metabolites concentration
The results of the main and interaction effects of different treatments are presented in Table 2. Based on the results, each of the measured factors has been interpreted and reviewed.

Phenol content
As it is shown in figure 9 A, among treatments in unrooted cuttings, the highest total phenol content Fig. 6 The mean values of rooting performance in rooted cut tings (percentage of rooting) within substrates and for the 5 treatments of indole butyric acid. The mean values with the same letter were not different at level 0.05 according to the LSD test. Sample data: 540 cuttings for each substrate.    Table 2 Results of a multifactor ANOVA used to examine the effect of major factors on the secondary metabolite composition of stem cuttings over four seasons In the table, the pvalues for the three principal effects (substrate, treatment with IBA and season, and their twoway interactions) are represented. Effects were considered significant when p<0.05. 540 stem cuttings in each season were planted.* p<0.5. (McDonald et al., 2001) was observed with no signifi cant difference in the fresh sample as well as in 4000 ppm and 8000 ppm of indole butyric acid treatments, and the lowest level was observed in control, 1000 ppm, and 2000 ppm treatments without any signifi cant difference. A fresh sample was prepared with other cuttings at the beginning of each season and is used only to measure the internal composition of the plant at the beginning of the season; no treatment is performed on it. It was to compare the amount of internal compounds of the plant at the beginning of the cutting time and compare it with the amount of these compounds after maintaining the cuttings in the substrate to root (control is a sample that is placed in the substrate without adding any treatment and is used to compare the effect of the treatments used on cuttings). Among the different substrates, the lowest amount of phenol content was found in stem cuttings that were planted in the mixed rooting substrate (Fig.  9 B). Among the unrooted cuttings, the lowest phe nol content was observed in a fresh sample and a treatment of 1000 ppm in January (Fig. 9 C). Between rooted cuttings, in April, with the highest rooting per centage of cuttings, treatments of 4000 and 8000 ppm showed lower phenol content, and there was no significant difference between other treatments (Fig.  9 D).

Flavonoid content
Among the unrooted cuttings in different seasons, the highest flavonoid levels were observed in January and July, and the lowest were seen in April and October (Fig. 10 A). The flavonoid content of Juniperus sabina differed during different seasons.
In rooted cuttings, flavonoid content was not sig nificantly different in treatments applied in different months, and the overall amount of flavonoid was between 50 and 100 mg/g of fresh weight (Fig. 10 B).

Percentage of antioxidant
In unrooted cuttings, the highest percentage of antioxidants was found in January and October with more than 70%, and the lowest was observed in July with a maximum of 20%. In April, an intermediate level of antioxidants was observed in unrooted cut tings (Fig. 11 A). In all months except July, the per centage of antioxidants in the first samples was about 70%, but in July it was about 20%. It should be noted that in July and January, the percentage of antioxidants increased after treating and planting the cuttings in substrate; this amount was unchanged in October (during fall) and it decreased in April (during spring), and its decline was also significant.
In rooted cuttings, the percentage of antioxidants in January was much higher April (Fig. 11 B). Among  different substrates, the lowest percentage of antiox idants in rooted cuttings was seen in mixed rooting substrate, and its maximum was seen in perlite sub strate (Fig. 11 C). Pumice and perlitecocopeat sub strate showed a medium antioxidant percentage. It means in the lighter substrate, the antioxidant per centage was increased, and in the heavier substrate, the percentage of that was decreased.

Discussion and Conclusions
Indole butyric acid is widely used at commercial level to root many species (Hartmann et al., 1990;Negash, 2002;Esmael Nia et al., 2006;Khoshnevis et al., 2012). It slowly releases a source of indole acetic acid (Epstein and LudwigMuller, 1993). Current evi dence indicates that indole butyric acid is naturally occurring in plants. Further stability of IBA in compar ison with indole acetic acid during rooting experi ments has been reported by Nordstrom et al. (1991), which is effective on decomposition and building. Part of the function of indole butyric acid is the direct effect of auxin (LudwigMuller, 2000;Poupart and Waddell, 2000). Although other functions are due to the conversion of IBA to IAA by boxidation (Epstein and Lavee, 1984;Zolman et al., 2000;Bartel et al., 2001).
Auxin can be increased for up to 24 hours after sampling (Tartoura et al., 2004;Osterc et al., 2009). Increasing root numbers after the use of indole butyric acid occurs in many woody plants (Jarvis, 1986). Adventitious roots on the cuttings were creat ed by treating them with auxin growth regulators, especially indole butyric acid (Buchala and Schmid, 1979;Haissig et al., 1992). This is consistent with the results of this research on its effectiveness on root ing. One possible explanation is that exogenous aux ins can increase the amount of internal auxin in the direction of the onset of the formation phase of the rooting and then the root appearance (Metaxas et al., 2004). With an increase in the presence of the cuttings in the substrate, the rooting rate of the cut tings also increases (Cope and Rupp, 2013). The use of indole butyric acid leads to an increase in rooting (Bielenin, 2003).
In our study, the best results were obtained from intermediate levels of IBA (10004000 ppm) without a significant difference between these treatments, and we hypothesize that IBA at 8000 ppm can dam age the cuttings and reduce rooting. In J. virginiana, IBA concentrations up to 2000 ppm did not stimulate rooting beyond that obtained with 5000 ppm (Henry et al., 1992). In general, IBA has been used for the rooting of Juniperus species with different treatment levels. For example, results were best at 8000 ppm of IBA in Juniperus osteosperma (Cope and Rupp, 2013), 5000 ppm of IBA in J. virginiana (Henry et al., 1992), 1000 ppm to 9000 ppm in Juniperus scopulorum (Bielenin, 2003), Chowdhuri (2017), with 1000 to 3000 ppm in Juniperus chinensis and Tektas et al., (2017) with 6000 ppm in Juniperus L. In the research on Juniperus virginiana, Henry et al. (1992) cited that in preliminary studies, IBA concentrations up to 20000 ppm did not stimulate rooting beyond that obtained with 5000 ppm. Thus, our results are more in agreement with Rifaki et al. (2002), which pro posed a concentration of 4000 ppm of IBA in cuttings of Juniperus excelsa, and Esmaeil Nia et al. (2006), with 3000 to 6000 ppm in J. excelsa. Nevertheless, the novelty of our results is that the concentration of IBA we selected (1000 ppm) was lower.
Substrate characteristics are very important in rooting success. Several studies have shown that the substrate plays a significant role in the quality of root formation and the percentage of rooted cuttings. Proper air preservation is a necessary feature of a good rooting atmosphere. Therefore, it seems that proper rooting substrate can maintain proper mois ture to prevent the cutting ends from drying out and to provide enough air to facilitate rooting and pre vent disease spread at the base of the cuttings. Surely there is an optimum temperature for sub strate for root formation and growth, and rooting at low temperatures will not occur or will occur very slowly. It is also possible for the roots to appear and grow at very high temperatures in the substrate. Bottomheat is useful for rooting only when the tem perature is low (Couvillon, 1988) which is consistent with the results of this research. In our study, the percentage of rooting was more than 60% in sub strate of perlite cocopeat. In an study of Juniperus procumbens the best substrate was 1.3 (v/v) vermi culite and 2.3 (v/v) perlite, with only 36% rooting (Hongwei et al., 2011). The results of our study was also better than the results obtained from Cuevas Cruz et al. (2015)  Cutting time plays an important role in the suc cess of rooting. Although many species are most rooted when cuttings are prepared in late spring or early winter before the wood has hardened, many other species have the best rooting when cuttings are taken at other times of the year. A good example of this is the Juniperus horizontalis, whose cuttings were most rooted when they were prepared between November and February compared to other times of the year (Ali Ahmad Koruri et al., 2011). The result of this research showed that the best time for rooting Juniperus sabina to prepare the cuttings is April. Therefore, for this species, the best time to prepare cuttings and plant them is spring. This differs from GuerreroCampo et al. (2006), who found the best rooting of several species of cuttings at different sea sons and Chowdhuri (2017), who showed the best rooting time for Juniperus chinensis was summer. On the other hand, our result was in agreement with Fragoso et al. (2015) and Tektas et al. (2017), who respectively cited the best season for rooting of Juniperus chinensis and Juniperus L. as spring.
Apparently, the presence of secondary metabo lites in plants acts as a defense (toxic) agent that inhibits proliferation and other growthrelated actions (Singh Rattan, 2010), as shown in the results of this study. Although most of the phenolic com pounds have a structural role in the cell wall, the major activity of these compounds is in defense of the plant; they have several roles in plants, but are mainly used for their great effects on growth, devel opment, propagation, as well as plant defense against animals and pathogens (Croteau et al., 2000). The presence and yield of secondary metabolites in plants, such as aromatic compounds and compounds in essential oils, may be affected in different ways, from formation to separation from plants. Rapid sec ondary metabolite induction occurs as a chemical mediator of plant rooting and defense (Metlen et al., 2009), and the amount of secondary metabolites changed during the preservation of cuttings in the substrate. The rooting barrier of yew cuttings was identified by biological and organic methods. The results showed that the most important barrier to propagation in this plant was phenol content (Guangyou, 2000).
The maximum amount of total phenol in the leaves of common juniper was 315.33 mg/g (Ved et al., 2017), which is consistent with the results of this study. In cherry leaf cuttings, GiSelA 5, auxin had no effect on phenol levels, so the same results were observed in the present study. Cuttings should have definite levels of different phenolic compounds to start the rooting induction phase, but the greater effect on rooting success is attributed to the effect of auxin level (Trobec et al., 2004).
Phenolic compounds are a class of antioxidants (Choudhury et al., 2013), and the level of internal antioxidants in plants is different (Rehman et al., 2014). Many authors have reported an association between total phenol content and antioxidant activi ty (Hariprasath et al., 2015). The main antioxidant activity is due to specific secondary metabolites, especially phenolic compounds and some terpenes (Marzouk et al., 2007;Awaad and AlJaber, 2010).
Interactions among genotypes, propagation meth ods, and growing seasons significantly affect flavonoid content and antioxidant capacity (Goyali et al., 2013), which is consistent with the results of this study. The amount of secondary compounds varied according to season and substrate, just as it did in the current study. The climate of the outdoor region during the three months of October, January, and April increased the amount of antioxidants inside the plant, while in July, with a hot climate, it dropped dramatically. Growth regulators increase antioxidant activity (Dakah et al., 2013), which contradicts the results of this research. Because in some cuttings treated with indole butyric acid, an increase in antioxidants was observed, and in other treatments, a decrease was observed.
The results of a study on one of the Iranian conifers showed that the antioxidant activity of the extracts ranged between 60 and 99% (Hariprasath et al., 2015), which contradicts the result of the present study, which shows that the range of antioxidants in some treatments was less than 20%.
In the use of indole butyric acid for the propaga tion of Juniperus sabina through cuttings, the best rooting month (season) for cuttings was April, and the rooting percentage in this month was higher than in other months (more than 50%), while instead, the lowest rooting rate was seen in January. The best lev els of indole butyric acid used were levels of 4000 and 1000 ppm, respectively. So, for the propagation of Sabina species, it is recommended to use these levels of IBA as a treatment for stem cuttings in April. Also, the best substrate used was perlitecocopeat. Between rooted cuttings, in April, with the highest rooting percentage of cuttings, treatments of 4000 and 8000 ppm showed lower phenol content; flavonoid content was not significantly different in treatments applied in different months and the per centage of antioxidants in January was much higher than April.