STUDY OF INDOLE BUTYRIC ACID ( IBA ) EFFECTS ON CUTTING ROOTING IMPROVING SOME OF WILD GENOTYPES OF DAMASK ROSES ( Rosa damascena Mill . )

Rosa damascena is very important for essential oil production, medicinal properties and it is also widely cultivated as a garden rose. The Rose species is mainly propagated by stem cutting. In the present study, the effect of different levels of 0, 500 and 1,000 mg l (quick dip method for 20 s) of indole butyric acid (IBA) on the rooting of 12 wild genotypes (including: Kurdistan 1 to Kurdistan 12) of R. damascena was investigated. The results show that the rooting ability of R. damascena differs significantly between the twelve genotypes. The highest rooting (79.56%) and callus production (69.08%), number of roots (8.33), root fresh and dry weights (361.80 and 244.74 mg, respectively) were recorded in Kurdistan 5 genotype with 1,000 mg l IBA. The maximum root length (5.84 cm) was observed in Kurdistan 5 genotype with 500 mg l IBA that showed a significant difference compared to the control treatment (0.96 cm). The highest number of leaves per bud (7.33 at 500 mg l IBA) and number of buds (5.00 at 1,000 mg l IBA) were recorded in Kurdistan 1 genotype. The current study demonstrated that the different genotypes of R. damascena were in a difficult-toroot state, which suggests that cutting treatment with 1,000 mg l IBA overcame the problem of the difficult-to-root state, and it can also enhance the rooting percentage in the studied genotypes.


Introduction
Rose is the most ancient ornamental species.There is evidence that roses were cultivated 5,000 years ago in China, western Asia, and northern Africa (Gudin, 2001).The genus Rosa, belonging to the Rosaceae family, includes 200 species and more than 18,000 cultivars (Gudin, 2003).One of the most important Rosa species is Rosa damascena Mill.Some of its varieties are very important for essential oil production and others are widely cultivated as garden roses (Rusanov et al., 2005).Damask roses are well known for their strong fragrance (Widrlechner, 1981).Rosa damascena is also cultivated for its medicinal properties and this aspect is steadily increasing in the world (Tabaei-Aghdaei et al., 2007).Iran represents a center of genetic diversity of the Damask roses (Babaei et al., 2007;Tabaei-Aghdaei et al., 2007;Kiani et al., 2008).
The Damask rose is commonly propagated by asexual methods (sucker, hardwood cutting, semi-hardwood cutting, budding and grafting) (Hartmann et al., 2002).Vegetative propagation is the sole method to maintain desirable characters in a superior cultivar particularly when it is heterozygous and polyploid.The use of stem cuttings is the easiest and the most common method of growing roses (Anderson and Woods, 1999).The success of rooting in cuttings depends upon the species and cultivar, condition of cutting wood, type of cuttings (hardwood, semihard wood cuttings, softwood and herbal cuttings), season and many other factors (Hartmann et al., 2002;Daneh-loueipour et al., 2006).
Auxin is widely used on the stem cuttings for accelerating the formation of adventitious roots (Galavi et al., 2013).Auxin has an effect on speed and increases the percentage of rooting of the stem cuttings (Kasim and Rayya, 2009).Auxins that have been found most reliable in stimulating adventitious root production in cuttings include indole acetic acid (IAA), naphthalene acetic acid (NAA) and indole butyric acid (IBA) (Randhawa and Mukhopadhyay, 1994).IBA is the most effective on promoting root-initiation and adventitiousroot production in stem cuttings (Waisel, 1991).The first adventitious roots appear from callus and they are main roots for cuttings.Callus contains a high amount of auxins (Hartman et al. 2002).Ercisli et al. (2001) studied the effects of IBA on adventitious root formation of wood cuttings in rose, as well as which IBA is suitable for rotting (Hartmann et al., 2002).In Bougainvillea, Hibiscus and Keiapple, the same advantage has been shown (Mudge et al., 1995).Rooting capacity for stem cuttings will be determined by the interaction of hereditary factors in stem cells and the following factors: auxin level, leaves and buds on the cuttings, the amount of carbohydrate reservoir in the cuttings, stage of plant growth, stem location and a type of the cutting tissue (Rosier et al., 2006).Adventitious root formation is regulated by complex interactions between endogenous and exogenous factors which affect the various developmental stages of root formation.In general terms, adventitious root formation follows three developmental phases: (1) dedifferentiation, in which predetermined cells switch from their normal morphogenetic path to act as mother cells for the root primordia; (2) initiation, in which these cells start to divide and form the distinct structure of a root primordium; (3) elongation, during which the newly formed primordia form vascular connections and later protrude through the surrounding tissues to form roots (Hartmann et al., 2002).Many physiological studies have shown that auxin plays a central role in the developmental process of root initiation (Jarvis, 1986).The promoting effect of IBA on rooting is mainly the result of its conversion to IAA in plant tissue.However, IAA, which is needed for the rooting process, is oxidized readily in the plant by peroxidases, whereas IAA released from IBA is not oxidized by peroxidases and remains at the base of the cutting (Abu-Zahra et al., 2012).
The aim of the present study was to find a practical method to promote rooting of Damask rose cuttings by IBA application.

Material and Methods
The experiment was carried out in the Department of Horticultural Science, Agriculture Faculty, University of Lorestan, Iran.Uniform hardwood cuttings of 12 wild genotypes (Kurdistan1 to Kurdistan 12) of Rosa damascena Mill.with at least 4 nodes and 8−10 mm in diameter and 20−22.6 cm in length were selected from the middle portion of the vigorously growing shoots in the winter while a plant was in the dormant phase.
The cuttings were disinfected by 3% sodium hypochloride for 5 seconds and then were washed with sterile distilled water.The basal portion of cuttings (3.0 cm) was treated with IBA (dissolved in 1.5 % (V/V) aqueous ethanol) at the rate of 0, 500 and 1,000 mg.l -1 (quick dip method for 20 s), and then were cultured in the growth medium.Control cuttings were dipped in 1.5 % (V/V) aqueous ethanol for the same time.The cuttings were planted at depth of 3 cm on planting medium in the greenhouse equipped with an intermittent mist system (scheduled for two-minute spray every 45 minutes from 7:00 A.M. to 8:00 P.M. daily and twice operation around midnight to maintain (Izadi and Zarei, 2014) an average relative humidity of the greenhouse in 70% ± 5%) (Izadi and Zarei, 2014).The environment temperature of cuttings was 22−24 °C and the bed temperature was 24−27 °C.The cuttings were rooted under a 16-h photoperiod.Growth bed contained sand-perlite and peat moss.After 75 days, traits such as root diameter (mm), root length (cm), number of roots, percentage of rooting, percentage of callusing, diameter of bud (mm), length of bud (mm), number of buds, number of leaflets per leaf, number of leaves per bud and root fresh weight (mg) and root dry weight (mg) were recorded.After calculating root fresh weight, they were wrapped in aluminum foil and dried in the oven at 80°C for 24 hours to calculate dry weight.
The experiment was conducted in a factorial design in a randomized complete block design in greenhouse conditions with 3 replicates, each replicate consisted of 25 cuttings and totally, seventy-five cuttings were used for each treatment.The data were analyzed using SAS software and means were compared through Duncan's test (p < 0.05).

Results and Discussion
The effect of different IBA concentrations on measured traits in Rosa damascena is shown in Table 1.IBA treatment had a significant influence on rooting and callusing percents (p<0.5).Among the genotypes tested, the highest rooting (79.56%) and callusing (69.08%) percents were recorded in Kurdistan 5 genotype with 1,000 mg l -1 IBA that made a significant difference compared to 500 mg l -1 IBA (with 61.15% of rooting and 51.27% of callusing) and control (with 36.93% of rooting and 41.19% of callusing) treatments.Among the genotypes rooted with attentive to IBA effect (Table 1), the lowest rooting (24.67%) and callusing (24.92%) percents were recorded in the genotypes Kurdistan 10 with 500 mg l -1 IBA and Kurdistan 12 with 500 mg l -1 IBA, respectively (Table 1).
Increased rooting percentage in the genotypes Kurdistan 5 and Kurdistan 1 might be due to better availability of carbohydrates and nitrogen compounds in the cuttings at the time of their collection.Similar results were reported by Khan et al., (2004) in other rose species.
IBA at 1,000 mg l -1 concentration had the highest rooting percent in all tested genotypes.Without IBA application, except for Kurdistan 1, 2, 3, 4, 5 and Kurdistan 7, other genotypes had no rooting which indicated a high level of difficult-to-root genotypes.This may be because the internal auxin amount is not enough for root induction of cuttings.It has been reported that auxin existence is necessary for induction of the root starter cells (Hartman et al., 2002).The IBA effects on rooting were in accordance to the findings on Rosa canina (Kazankaya et al. (2005), on Syzygium javanica (Paul and Aditi, 2009), on five pomegranate varieties (Saed, 2010), on Parthenocissus quinquefolia (Abu-Zahra et al., 2012) and on Vitisvinifera (Galavi et al., 2013).The effects of different IBA concentrations on rooting ability of stem cuttings of Rosa damascena were also investigated previously by Ercisli et al. (2001), Akhtar et al. (2002), Khan et al. (2004), Kazankayaet al. (2005), Dawa et al. (2013) and it has been indicated that IBA had a significant effect on the rooting.Differences in rooting cuttings among genotypes might be due to variable endogenous auxin levels in cuttings and inherent genetic characteristics of genotypes.0.00 h 0.00 g 0.00 g 0.00 e 0.00 i 0.00 q 0.00 n 0× Kurdistan 8 0.00 h 0.00 g 0.00 g 0.00 e 0.00 i 0.00 q 0.00 n 0× Kurdistan 9 0.00 h 0.00 g 0.00 g 0.00 e 0.00 i 26.74 n 0.00 n 0× Kurdistan 10 0.00 h 0.00 g 0.00 g 0.00 e 0.00 i 0.00 q 0.00 n 0× Kurdistan 11 0.00 h 0.00 g 0.00 g 0.00 e 0.00 i 0.00 q 0.00 n 0× Kurdistan 12 0.00 h 0.00 g 0.00 g 0.00 e 0.00 i 0.00 q 0.00 n 1000× Kurdistan 0.00 h 0.00 g 0.00 g 0.00 e 0.00 i 0.00 q 0.00 n 1000× Kurdistan 13.58 gh 34.09 fg 0.43 efg 1.73 cde 2.00 fg 33.45 j 29.34 l 1000× Kurdistan 0.00 h 0.00 g 0.00 g 0.00 e 0.00 i 27.20 n 0.00 n 1000× Kurdistan 0.00 h 0.00 g 0.00 g 0.00 e 0.00 i 0.00 q 0.00 n 1000× Kurdistan Means in a column followed by the same letter are not significantly different at the 5% level as determined by Duncan's test.* R.F.W.: Root Fresh Weight, R.D.W.: Root Dry Weight.
Similar findings have been reported by Bharathy et al. (2004) in case of carnation and by Kazankaya et al. (2005) in case of other cultivars of rose.The exogenous application of IBA generally improves rooting in rose species (Akhtar et al., 2002;Khan et al., 2004 andDawa et al., 2013).A quick dip in auxin solutions might have supplemented the endogenous auxin content at the base of cuttings, which accelerated the root initiation and formation of root primordia that resulted in increased rooting in treated cuttings.It is generally known that the formation of adventitious roots in plants is controlled by growth substances and auxins are the principal hormones playing a direct role in this process (Gaspar and Hofinger, 1988).According to Sebanek et al. (1991), the formation of adventitious roots is related to an increase in the level of auxin at stem base.Auxins can control meristematic activity of tissues and also enhance the supply of plastic substances at the sites of root formation.This confirms that IBA is one of the widely applicable root-forming stimulants.IBA is known to induces a high number of adventitious roots (Hartmann et al., 2002).It has been proved as the most efficient treatment and it additionally induces earlier root formation (Mateja et al., 2005).Naier et al. (2008) investigated the effect of auxin concentration on the rooting of Stewartia pseudocamellia and announced that cuttings treated with rooting hormones had higher rooting percentages (71.9% to 93.6%) as compared with the control (53%).
According to Hartmann et al. (2002), IBA is the best root promoter due to its fast auxin activity and an enzymatic system of fairly slow destruction.Strydom and Hartman (1959) found the positive effect of auxin on the increase of respiration rate and a high level of amino acid storage at the base of cuttings 24 hours after treatment with auxin.
This study showed that the Damask rose cuttings in rooting had little potential and the use of auxins had a significant effect on rooting percentage (Table 1, Figure 1).For successful rooting induction, plants should contain a certain quantity of IBA (Sulusoglu and Cavusoglu, 2010).The application of IBA may have an indirect influence by enhancing the speed of transformation and movement of sugar to the base of cuttings and consequently rooting.Obtained results (Table 1, 2) are in conformity with the results of Pandey et al. (2011).Kumari et al. (2010) reported that auxins can control cell enlargement, bud formation and root initiation and also promote the production of other hormones.
Results showed that the number of roots, root length, and root diameter were significantly affected by IBA treatment at 5% level (Table 1).Significant variations in root number and length were observed in the evaluated genotypes.The maximum number of roots (8.33) and length (5.84 cm) were observed in Kurdistan 5 genotype with 1,000 and 500 mg.l -1 IBA, respectively, that showed a significant difference at 5% level compared to the control (with number of 1.00 of root and 0.96 cm in length) treatment (Table 1, Figure 1).Among the genotypes rooted with IBA, the minimum number of roots (1.69) and length of root were obtained in Kurdistan 10 genotype with 500 mg.l -1 IBA.
A significant variation in the primary root number and length among the cultivars may be due to a genetic component or difference in the endogenous auxin level of the cuttings.Similar findings were reported by Akhtar et al. (2002) on Rosa centifolia and Rosa damascena and by Khan et al. (2004) on different rose rootstocks.In the present study, IBA significantly improved the number of primary roots per cutting and root length (Table 1, Figure 1).These results are in close k l m conformity with findings on scented geranium (Shukla et al. 2004) and Syzygium javanica (Paul and Aditi, 2009).
The highest diameter of root (1.36 mm) was obtained in Kurdistan 5 genotype with 1,000 IBA mg l -1 that showed a significant difference at 5% level compared to 500 mg l -1 of IBA (0.90 mm) and the control (0.34 mm) treatments.All rooted cuttings under treatment with IBA showed thicker roots than the control treatment.Bhatt and Tomar (2010) have believed that root diameter increased by increased auxin levels.This phenomenon might be attributed to greater metabolic activity and maximum utilization of sugar and starch after hydrolysis of stem (Bhatt and Tomar, 2010).In an experiment, increased concentration in IBA from 1,000 to 2,000 mg l -1 increased the root length, number of roots, root fresh weight and root dry weight in cuttings (Rahdari et al., 2010).
Results showed that IBA had a significant influence on root fresh and dry weights at 5% level (Table 1).Among the tested genotypes, the highest root fresh and dry weights (361.80 and 244.74 mg, respectively) were recorded in Kurdistan 5 genotype with 1,000 mg l -1 IBA that showed a significant difference compared to 500 mg l -1 IBA (with 92.38 and 72.63 mg of root fresh weight and root dry weight, respectively) and the control (with 9.15 and 5.21 mg of root fresh weight and root dry weight, respectively) treatments.Among the rooted genotypes with IBA, the lowest root fresh and dry weights (9.75 and 5.88 mg respectively) were recorded in Kurdistan 1 genotype with 500 mg l -1 IBA (Table 1).Our results were similar with those reported by Al-Salem and Karam (2001) on Arbutus andrachne.Simultaneously, stimulation of rooting process with auxin, carbohydrate transportation from leaf to root increases, therefore, it causes increasing of dry weight of root (Hartmann et al., 2002).Also, Karimi (2011) in his experiment about cutting-grafting of Punica granatum showed that the cutting-grafting treated with IBA had a higher root fresh weight than the control.
IBA significantly increased the number of new leaves in rooted cuttings (Table 2).Among the tested genotypes, the highest number of leaves per bud (7.33 at 500 mg l -1 IBA) and number of buds (5.00 at 1000 mg l -1 IBA) were recorded in Kurdistan 1 genotype that showed a significant difference compared to the control (with 2.33 and 2.33 leaves per bud and buds, respectively) treatment (Table 2).Increase in the new leaf production in cuttings might be attributed to an increased root number and root length in growth regulator treated cuttings that might have enabled cuttings to absorb more water and nutrients from rooting media, leading to better growth and production of new leaves.These results are in close agreement with the findings of Ingle and Venugopal (2009) in case of Stevia and Saed (2010) in case of five varieties of pomegranate.Also, differences among cultivars with respect to the emergence of new leaves might be due to variation in their growth rate, which is generally controlled by genetic factors.Since root is considered as main source of cytokinin, it may help producing higher leaf number.Moreover, leaf is a source of auxin and carbohydrate.Carbohydrates have been considered optimal markers since they are the main energetic resource during the rooting process.Auxin and carbohydrate produced in leaf move to portion base of the cutting.It helps of interaction of endogenous IAA with exogenously applied IBA in the base of the cutting for the emergence of root primordial (Hartman et al., 2002).Hence, with an increasing number of leaves, cuttings higher adventitious roots (Shukla et al., 2004); when cuttings had higher roots, the weight of adventitious roots was greater.The relationship between the number of leaves and the weight of adventitious roots of cuttings of R. damascena agrees with results of different R. canina 'Inermis' clones (de Vries and Dubois, 1987b), Ficus (Poole and Conover, 1984), and R. hybrida 'Sonia' (Dubois and de Vries, 1985).
Among the tested genotypes, the highest length of bud and diameter of bud (37.80 and 4.91 mm, respectively) were recorded in Kurdistan 1 and Kurdistan 3 genotypes, respectively with 1,000 mg l -1 IBA that showed no significant difference compared to 500 mg l -1 IBA (with 31.96mm and 4.72 mm in length and diameter of bud, respectively), but showed a significant difference compared to the control (with 16.24 mm and 3.60 mm in length and diameter of bud, respectively) treatment (Table 2).As roots are important sources of cytokinins which are known to promote bud-break in roses (Mor and Zieslin, 1987), cytokinin production in young root tips might be an important cause of the sprouting of axillary buds.
These results show that the rooting ability of R. damascena differs significantly between the twelve genotypes and that a high dose of IBA induced a better rooting.Indeed, in practice of the cuttings, meristem initiation is generally favored by a relatively high input of exogenous auxin concentration (Margara, 1979).

Conclusion
The current study demonstrated that the different genotypes of R. damascena were in a difficult-to-root state, which suggests that a cutting treatment with 1,000 mg l -1 IBA overcame the problem of difficult-to-root state.

Table 1 .
Effects of IBA on rooting of Damask rose (Rosa damascena).

Table 2 .
Effects of IBA on rooting of Damask rose (Rosa damascena).