Impaginato 105 Adv. Hort. Sci., 2019 33(1): 105-111 DOI: 10.13128/ahs-22596 Propagation of Rosa hybrida L. cv. Dolce Vita by stenting and stem cutting methods in response to different concentrations of IBA M. Pourghorban 1, S. Khaghani 1 (*), P. Azadi 2, 3 (*), A. Mirzakhani 4, M. Changizi 1 1 Department of Agriculture, Arak Branch, Islamic Azad University, Arak, Iran. 2 Department of Genetic Engineering, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), P.O. Box 31535-1897 Karaj, Iran. 3 Department of Tissue Culture, Ornamental Plants Research Center, Horticultural Science Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Mahallat, Iran. 4 Horticulture Crops Research Department, Markazi Agricultural and Natural Resources Research and Education Center, AREEO, Arak, Iran. Key words: Indole-3-butyric acid (IBA), propagation, rooting, rootstock, stenting. Abstract: This study was conducted to investigate the effect of different con- centrations of Indole-3-butyric acid (IBA) in propagation of Rosa hybrida L. cul- tivar Dolce Vita by stenting (cutting and grafting) and stem cutting methods in greenhouse conditions. Different concentrations of IBA (0, 1500, 3000 and 4500 mg/L) were considered on Rosa hybrida L. cv. Dolce Vita grafted onto Rosa hybrida L. ‘Natal Briar’ rootstock. Then, the stentings and stem cuttings were cultured in a cocopeat + perlite (in 1:1 ratio) medium under mist system. The research was conducted as a completely randomized design with three replica- tions. The results suggested that all IBA treatments significantly increase root- ing percentage compared with the control plants, and the highest specifications of roots and shoots were observed in 1500 mg/L IBA in both methods of propa- gation. The results approved the superiority of stem cutting in rooting. However, the higher content of chlorophyll a, b and total were obtained in stenting method. 1. Introduction Rose flower (Rosa hybrida L.) is one of the world’s most popular flow- ers among ornamental plants (Castilon et al., 2006), and cut rose flower industry is the most important aspect of rose culture industry in the world (Bleeksma and Van Doorn, 2003) with a turnover of 735 million Euros in 2015 (Azadi et al., 2016). Rose plants are propagated by seed and asexual methods such as stem cutting, grafting, budding, cutting-grafting (stent- ing), cutting-budding, root grafting and tissue culture (Salehi and Khosh- (*) Corresponding author: azadip22@gmail.com azadip@abrii.ac.ir shahab.khaghani@gmail.com Citation: POURGHORBAN M., KHAGHANI S., AZADI P., MIR- ZAKHANI A., CHANGIZI M., 2019 - Propagation of Rosa hybrida L. cv. Dolce Vita by stenting and stem cutting methods in response to different concentrations of IBA. - Adv. Hort. Sci., 33(1): 105-111 Copyright: © 2019 Pourghorban M., Khaghani S., Azadi P., Mirzakhani A., Changizi M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 4 February 2018 Accepted for publication 30 November 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2019 33(1): 105-111 106 Khui, 1997 a, b; Dole and Wilkins, 2005; Azadi et al., 2007, 2013). Vegetative propagation is the sole method for maintaining desirable characters in a superior cultivar particularly when it is heterozygous and polyploid. The easiest and most common method of growing roses is the use of stem cuttings (Anderson and Woods, 1999). The success of rooting in stem cuttings depends on species and cultivar, growth season, the condition of cutting wood, type of cuttings like hardwood cuttings, semi hard wood cuttings, softwood and herbal cuttings, and many other factors (Hartmann et al., 2002). However, stenting method is an efficient technique for quick propagation of plants. This method is an effective technique of rose propagation (Van de Pol and Breukelaar, 1982), in which cutting and grafting is performed simultaneously and the scion is grafted onto a non-rooted rootstock. Formation of the union and adventitious roots on the rootstock occurs simul- taneously (Nazari et al., 2009; Karimi, 2011; Babaie et al., 2014). Cutting and grafting is also a valuable tech- nique in propagation of several horticultural species including roses (Koepke and Dhingra, 2013), conifers, rhododendrons, and also a number of citrus fruits, apples, plums and pears (Hartmann et al., 2002), therefore the use of rootstocks in propagation is a common practice. Plant propagation by stenting has been practiced in several nurseries as a substitute for budding, since it has advantages such as better yield and quality, and resistance to crown gall disease (Park and Jeong, 2010). Efficiency and flower produc- tivity in grafted plants is higher than those in plants growing on their own roots and this is due to the use of rootstock (Cabrera, 2002). Because of different cli- matic and soil conditions in different areas of the world, different rootstocks are recommended to be the main subject of attention of compatibility between rootstock and scion (Niu and Rodriguez, 2008). ‘Natal Briar’ is a rootstock of unknown lineage, probably coming from South Africa, and is becoming the desirable rootstock for hydroponic production systems in Netherlands, USA and Colombia (Cabrera, 2002). ‘Natal Briar’ is the rootstock for grafted roses, which represents 60-70% of the world’s cultivation since it is easy to root and gives well stem length and head size (Otiende et al., 2015). Exogenous auxin is shown to have a necessary role in root formation in cuttings and has an effect on the speed and the per- centage of rooting of cuttings. Plants produce natural auxin in their fresh shoots and leaves, however, the synthetic auxin must be used for successful rooting to prevent cuttings death (Stefanic et al., 2006; Kasim and Rayya, 2009), and has been reported in many species, e.g. Hibiscus rosa sinensis (Kumar and Singh, 2012). For accelerating the formation of adventitious roots, auxin is widely used on the stem cuttings (Galavi et al., 2013), and it increases the speed and percentage of rooting (Kasim and Rayya, 2009). Photosynthetic pigments in plants comprise chloro- phylls a and b and these pigments play an important role in light absorption during photosynthesis (Lobato et al., 2009). Carotenoids are a group of nat- ural tetraterpenoid pigments distributed widely in plants, algae, fungi, and bacteria. Many flowers, fruits, and roots owe their vivid orange, yellow, and red colors to carotenoids. Carotenoids play necessary roles in photosynthesis and photoprotection (Domonkos et al., 2013; Niyogi and Truong, 2013; Hashimoto et al., 2016). This research was carried out to evaluate different concentrations of IBA in propagation of Rosa hybrida L. cultivar ‘Dolce Vita’ using stem cutting and stenting. 2. Materials and Methods Greenhouse conditions This research was carried out in February 2017 in a greenhouse with plastic cover located in Markazi Province, Arak, Iran. The greenhouse was equipped with mist system (Fig. 1 a) as well as hot water sys- tem for adjusting temperature and humidity. During the study cycle, the average temperature and rela- tive humidity of day were maintained at 20±5°C and 85±5%, respectively. At night, the mist system was automatically switched off and temperature was maintained at 10-15°C and relative humidity of 65±5%. Plant materials and propagation (stenting and stem cutting) Plant materials were prepared from a Rose com- mercial greenhouse (located in Markazi Province, Arak, Iran). The scions consisted of a single-node and one leaf (including 2 leaflets) were collected from Rosa hybrida L. cv. Dolce Vita as soon as their mother plants entered to faded flower stage. The scions were grafted onto a 4.0 cm length internode taken from the semi-hard wood cuttings of the rootstock Rosa hybrida L. ‘Natal Briar’. In this method, scions and rootstocks with a suitable flat cut could be graft- ed together with the maximum overlap of the cambi- um layer (Fig. 1 b). The scions were selected based on thickness of the stem rootstock. Scions then were Pourghorban et al. - Propagation in Rose 107 grafted using splice grafting method. Scions and rootstocks with an appropriate smooth cut could be grafted together with the maximum overlap of the cambium layer. Plastic tape was used for wrapping the graft union. The end bud in the rootstock was removed for better rooting and avoiding sucker pro- duction (Fig. 1 c). Stem cuttings with at least 3 nodes and of 5-8 mm diameter and 15-20 cm length were selected from the middle portion of the vigorously growing shoots of Rosa hybrida L. cv. Dolce Vita as soon as their mother plants entered to faded flower stage. The end node of the stem cuttings were removed for better rooting. The distal end of root- stocks and stem cuttings were treated with different concentrations of IBA (1500, 3000 and 4500 mg/L) by quick dip method, and control plants were treated with distilled water. The stentings and stem cuttings were rooted in a mixture of cocopeat and perlite medium (in 1:1 ratio) under mist system. To prevent fungal infection, the beds were disinfected with fungicides 0.2% ‘Captan’ solution every two weeks. The grafted plants and stem cuttings were grown for 60 days under the mentioned greenhouse condi- tions. At the end of the study, the plants were removed from the culture medium and some of their morphological and biochemical traits including heal- ing percentage, rooting, root number, longest root, fresh weight of roots, dry weight of roots, shoot per- centage, leaf number, shoot number, longest shoot, content of chlorophyll (a, b and total) and carotenoid were measured. Chlorophyll (a, b and total) and carotenoid contents measurement procedure To measure the chlorophyll content, 0.2 g of fresh leaves were ground to a fine pulp by adding 10 ml of 80% acetone. The supernatant was transferred to a 25 ml volumetric flask. The washings were collected in the volumetric flask and the volume was made up to 25ml with 80% acetone. The solution was cen- trifuged (5000 rpm) for five minutes. The absorbance of the solution was read at 663, 645 and 470 nm against the solvent (80% acetone) blank (Lichtenthaler, 1987). Chl a (mg/ml) tissue = [12.7(A663) - 2.69 (A645)] V / 1000 W Chl b (mg/ml) tissue = [22.9(A645) - 4.68 (A663)] V / 1000 W Total Chl (mg/ml) tissue = [20.2(A645) + 8.02(A663)] V / 1000 W Car (mg/ml) tissue = [(1000 A470 - 1.8 Chl a - 85.02 Chl b)/198] V / 1000 W where Chl= Chlorophyll, Chl a= Chlorophyll a, Chl b= Chlorophyll b, Car= Carotenoid, A= Absorbance at specific wavelengths, V= Final volume of chlorophyll extract in 80 % acetone (25 ml), and W= Fresh weight of tissue extracted (0.2 g). Data collection and statistical analysis Sixthy days after planting, the stentings and stem cuttings were taken out from the media and some traits such as rooting percentage, shoot number, root number, root length (cm), leaf number, shoot length (cm), contents of leaf chlorophyll (a, b and total) and carotenoid (mg/g), root fresh and dry weight (g), and healing (graft-take) percentage were recorded for each stenting and stem cutting. After calculating fresh weight of roots, they were wrapped in paper envelopes and dried in oven at 60°C for 24 hours to calculate their dry weight. Content of leaf chlorophyll and carotenoid were determined using spectropho- tometric method (Saini et al., 2001). The experiment was conducted as a completely randomized design with three replications, and each replication consist- ed of 16 samles. The data were analyzed with SAS software and means were compared through Duncan test (p<0.05). 3. Results The influence of treatments on morphological parameters and graft healing of stentings are shown Fig. 1 - Greenhouse space and the stages of preparation of stentings. Mist system in the greenhouse (a), Flat cut in Scions and root- stocks (b), Connection scions and rootstocks by plastic tape, and remove end bud (c). Adv. Hort. Sci., 2019 33(1): 105-111 108 in Table 1. The presented data clearly revealed that different concentrations of IBA significantly affected various traits of roots and shoots. Maximum rooting percentage, root number, average root length, and fresh and dry weight of roots were recorded in 1500 mg/L of IBA. The highest grafting success, number of leaves, number of shoots per stenting, and average shoot length were observed in 1500 mg/L of IBA con- centration (Table 1). An example of rooted stentings is shown in figure 2 (a-d). The influence of treatments on morphological parameters in stem cuttings is also shown in Table 2. Maximum rooting percentage, root number, and fresh and dry weight of roots in stem cuttings were recorded in 3000 mg/L of IBA. An example of rooted stem cuttings is shown in figure 3 (a-d). Different concentrations of IBA had significant effect on rooting percentage, root number, and fresh and dry weight of the roots. The highest rooting per- centage, root number, fresh and dry weight of roots was obtained in stem cuttings treated with 3000 mg/L of IBA. However, there was no significant dif- ference between treatments (1500 and 3000 mg/L) in most of the traits of stem cuttings (Table 2). Results showed that contents of leaf Chlorophyll (a, b and total) and carotenoid in stentings and stem cut- tings were affected by different concentrations of IBA (p<0.01). An example of rooted stentings is shown in figure 4 (a-d). Healing percentage in stentings The presented data clearly revealed that maxi- mum grafting success (66.66%) was in 1500 mg/L of IBA, and the minimum healing percentage was observed in control and 4500 mg/L of IBA (Table 1). Root indices and fresh and dry weight of roots The highest percentage of rooting were observed in stentings (81.25%) and stem cuttings (97.91%) treated with 1500 and 3000 mg/L of IBA, respective- ly, and the minimum rooting percentages (35.41% and 83.33%) in both propagation methods were Table 1 - The effect of IBA treatments on growth parameters and graft healing of Rosa hybrida L. cv. Dolce Vita stentings Fig. 3 - Rooting level at different concentrations IBA in Rosa hybrida L. cv. Dolce Vita stem cuttings. Control plant (a), Concentration of 1500 mg/L IBA (b), Concentration of 3000 mg/L IBA (c) and Concentration of 4500 mg/L IBA (d). Fig. 2 - Rooting level at different concentrations IBA in Rosa hybrida L. cv. Dolce Vita stentings. Control plant (a), Concentration of 1500 mg/L IBA (b), Concentration of 3000 mg/L IBA (c) and Concentration of 4500 mg/L IBA (d). IBA (mg/L) Healing (%) Rooting (%) Root number Longest root (cm) Roots fresh weight (g) Roots dry weight (g) Shoot (%) Leaf number Shoot number Longest shoot (cm) 0 37.50 b 35.41 b 1.96 b 0.63 c 0.25 c 0.02 c 37.5 b 1.25 b 0.44 b 2.22 b 1500 mg/L 66.66 a 81.25 a 9.60 a 3.00 a 0.95 a 0.12 a 66.66 a 2.50 a 0.70 a 5.32 a 3000 mg/L 43.75 b 50.00 b 4.54 b 1.93 b 0.60 b 0.07 b 43.75 b 1.96 ab 0.46 ab 3.51 b 4500 mg/L 37.50 b 41.66 b 3.73 b 1.71 b 0.51 b 0.06 b 37.50 b 1.59 b 0.37 b 2.80 b Table 2 - The effect of IBA treatments on growth parameters of Rosa hybrida L. cv. Dolce Vita stem cuttings IBA (mg/l) Rooting (%) Root number Longest root (cm) Roots fresh weight (g) Roots dry weight (g) Shoot (%) Leaf number Shoot number Longest shoot (cm) 0 83.33 b 13.95 d 5.06 b 1.23 d 0.12 c 93.75 a 4.8 a 1.69 a 7.87 b 1500 mg/L 95.83 a 22.94 b 8.21 a 1.76 b 0.16 b 95.83 a 4.83 a 1.61 ab 11.20 a 3000 mg/L 97.91 a 26.49 a 7.73 a 2.01 a 0.22 a 97.91 a 4.29 b 1.41 ab 10.46 a 4500 mg/L 85.41 b 20.72 c 5.80 b 1.55 b 0.15 bc 85.41 a 3.39 c 1.35 b 8.73 b Pourghorban et al. - Propagation in Rose 109 recorded in control plants (Table 1, 2). Based on this study, different concentrations of IBA affected root number in stentings and stem cuttings. The maxi- mum root number in stentings (9.60) and stem cut- tings (26.49) were observed in 1500 mg/L and 3000 mg/L of IBA, respectively (Table 1 and 2). Results showed the greatest average root length in stentings (3 cm) and stem cuttings (8.21 cm) on 1500 mg/L of IBA, and the smallest rooting length in stentings (0.63 cm) and stem cuttings (5.06 cm) were observed in control plants (Table 1 and 2). The greatest root fresh weights were found in 1500 and 3000 mg/L of IBA respectively in stentings (0.95 g) and stem cuttings (2.01 g), and the lightest root fresh weights in both propagation methods were observed in control plants. The greatest dry weight of the root in stent- ings (0.12 g) and stem cuttings (0.22 g) were also observed at 1500 and 3000 mg/L of IBA, respectively, and the lightest dry weight of the root in both propa- gation methods were recorded in the control plants (Table 1 and 2). Number of leaves, number and length of shoots In stentings, highest leaf number (2.50), shoot number (0.70) and shoot length (5.32 cm) were observed in 1500 mg/L of IBA. The lowest leaf num- ber (1.25) and shoot length (2.22 cm) were recorded in control plants, while the lowest shoot number (0.37) was observed in 4500 mg/L of IBA (Table 1). In stem cuttings, highest leaf number (4.83) and shoot length (11.20 cm) were observed in 1500 mg/L of IBA, while highest shoot number (1.69) was observed in control plants (Table 2). Contents of leaf Chlorophyll (a, b and total) and carotenoid In this study, contents of chlorophyll a, b and total and contents of carotenoid were measured in stent- ings and stem cuttings. In 0.2 g of fresh leaves, the highest content of chlorophyll a (1.03 and 0.69 mg/g), chlorophyll b (0.57 and 0.32 mg/g), total chlorophyll (1.62 and 1.03 mg/g) and carotenoid (0.77 and 0.49 mg/g) were observed in the 1500 mg/L of IBA in stentings and stem cuttings, respec- tively (Fig. 4 a-d). 4. Discussion and Conclusions Physiologically, stenting is more complicated than cutting propagation since formation of the graft union must occur simultaneously with rooting and there are interactions between photosynthesis, root formation, and bud development (van de Pol et al., 1986). Karimi (2011) also reported that in stenting, the graft union must be formed before root initia- tion. Therefore, after leaf formation on the scion, car- bohydrates and natural hormones are produced and transmitted from the leaves to the rootstock for growing. Another study reported that rootstock plays an important role in the entire process of root growth, and propagation through bench grafting will bring success. In propagation of Chinese hibiscus through stenting showed that IBA treatments signifi- cantly increased rooting percentage (Izadi and Zarei, 2014). The effect of different concentrations of auxin on rooting of Stewartia pseudocamellia, reported that rooting percentages in cuttings treated with rooting hormones (71.9% to 93.6%) are higher than rooting percentages(53%) in the control plants (Nair et al., 2008). For root formation on stem cuttings, natural or synthetic auxins are essential. Auxin increases the formation of adventitious roots in many species through facilitating carbohydrates and nitro- gen materials transfer to the cutting base and moti- vating primordial root. The high level of auxin may have a negative effect on root length. Auxin leads to transfer of leaf carbohydrate and nitrogen to the roots and therefore causes an increase in the root dry weight (Hartmann et al., 2002). Al-Salem and Karam (2001) reported that auxin concentration had a significant effect on rooting regardless of its chemi- cal structure or type. The maximum rooting percent- age, number of root, length, and fresh and dry weight were obtained by basal cuttings treatment with 24 mM of IBA. The maximum dry weight of roots may be Fig. 4 - Comparison content of chlorophyll and carotenoid (mg/ml) in Rosa hybrida L. cv. Dolce Vita stentings and stem cuttings treated with different concentrations of IBA (mg/L). Content of Chlorophyll a (a), content of Chlorophyll b (b), total chlorophyll content (c) and caro- tenoid content (d). 110 Adv. Hort. Sci., 2019 33(1): 105-111 ascribed to increased roots length and number of roots (Ingle and Venugopal, 2009). Auxins promote adventitious root formation and formed roots enhance the uptake of water and mineral nutrient and production of hormones (cytokinins) which are required for shoot growth and development (Otiende et al., 2015). Growth amount of shoots and roots are interdependent (Tonutti and Giulivo, 1990). The probable cause for increase in shoot length may be the better utilization of carbohydrates, nitrogen and other nutrients which has been assisted by growth regulators (Chandramouli, 2001). Izadi and Zarei (2014) reported that the highest leaf number was observed in stentings with higher root number. Buds and leaves are considered significant factors to improve root induction (Hartmann et al., 2002). Leaf chlorophyll content was affected by the interaction between cultivars and propagation methods and was significant in all grafted cultivars; as a result, leaf chlorophyll content and quality index were higher in grafted plants compared to those propagated by cut- tings. This might be the effect of rootstock (Nazari et al., 2009). In conclusion, the study demonstrated that the application of IBA plays an important role in suc- cess of Rosa hybrida L. cv. Dolce Vita propagation through stenting and stem cutting methods. 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