Acta agriculturae Slovenica, 119/1, 1–10, Ljubljana 2023 doi:10.14720/aas.2023.119.1.2380 Original research article / izvirni znanstveni članek Comparison of shoot and root regeneration of miniature potted rose (Rosa x hybrida L.) and Damask rose (R. damascena Mill.) in microcul- ture system Farkhondeh REZANEJAD 1, 2, Somayeh ABDIRAD 1, Moslem ABARIAN 1 Received October 16, 2021; accepted December 29, 2022. Delo je prispelo 16. oktobra 2021, sprejeto 29. decembera 2022 1 Department of Biology, Shahid Bahonar University of Kerman, Kerman, Iran 2 Corresponding author, e-mail: frezanejad @uk.ac.ir Comparison of shoot and root regeneration of miniature pot- ted rose (Rosa x hybrida L.) and Damask rose (R. damascena Mill.) in microculture system Abstract: Miniature potted rose and Damask rose are im- portant commercial plant cultivars in ornamental horticulture. Root suckers are common rose propagation method, but it is slow and seasonally dependent. In this survey, the propaga- tion of nodal explants of these two species was studied through in vitro regeneration system. 16 and 24 different media were used for study of shoot and root regeneration respectively. The axillary buds were sprouted earlier in miniature rose than R. damascena. Shoot induction and proliferation (shoot ramifica- tion and growth) were observed 5 and 17 days after planting in miniature rose and 16 and 38 days in R. damascena respectively. The highest shoot proliferation obtained in media 3 and 7 in miniature rose, and medium 16 for R. damascena. These three media were recorded as optimal media with 100 % shoot prolif- eration. In these media, root initiation and growth of miniature rose (respectively after 78 and 92 days) was earlier than Damask rose (respectively 125 and 138 days). The successful rooting oc- curred in three and two media for miniature and Damask rose respectively. Rooting frequency was higher in the half strength MS liquid media than the others. Thus, cultivar potted rose as a modern species is propagated easier than old rose (R. dama- scena). Key words: micropropagation; proliferation; rooting rate Primerjava regeneracije poganjkov in korenin pri miniaturni ločni vrtnici (Rosa hybrida L.) in damaščanski vrtnici (R. da- mascena Mill.) v mikrokulturi Izvleček: Miniaturna lončna vrtnica in damaščanska vr- tnica sta pomembni komercialni sorti med okrasnimi rastlina- mi. Za razmnoževanje vrtnic se najbolj pogosto uporabljajo po- ganjki iz korenin, a je njihova rast počasna in sezonsko odvisna. V raziskavi je bilo preučevano razmnoževanje teh dveh sort z izsečki nodijev v in vitro kulturah. Za regeneracijo poganjkov in korenin je bilo uporabljeno 16 in 24 različnih medijev. Zalistni brsti so odgnali prej pri miniaturni kot pri damaščanski vrtnici. Zasnova in rast poganjkov sta se pri miniaturni vrtnici pojavili 5 in 7 dni po sadnji in 16 ter 38 pri damaščanski vrtnici. Naj- večje število poganjkov je bilo pri miniaturni vrtnici v medijih 3 in 7 in v mediju 16 pri damaščanski vrtnici. Ti trije mediji so bili prepoznani kot optimalni, sa je bila v njih dosežena 100 % tvorba poganjkov. V teh medijih sta bili zasnova in rast korenin pri miniaturni vrtnici zgodnejši (po 78 in 92 dneh) kot pri da- maščanski vrtnici (po 125 in 138 dneh). Uspešno ukoreninjenje se je za miniaturno vrtnico pojavilo v treh medijih in le v dveh za damaščansko vrtnico. Pogostnost ukoreninjenja je bila večja v polovičnih tekočih MS medijih kot v drugih. Zaključimo lah- ko, da se miniaturna lončna vrtnica kot moderna vrsta razmno- žuje lažje kot starinska damaščanska vrtnica. Ključne besede: mikropropagacija; proliferacija; hitrost ukoreninjanja Acta agriculturae Slovenica, 119/1 – 20232 F. REZANEJAD et al. 1 INTRODUCTION There are more than 18,000 cultivars of roses, which collectively are based on only eight of the approximate- ly 200 wild species in Rosa: R. damascena, R. chinensis, R. wichuraiana Crép., R. odorata (Andrews) Sweet, R. moschata Herrm., R. multiflora Thunb., R. foetida Her- rm., and R. rugose Thunb.. The cultivation of roses for many purposes has been widespread in temperate cli- mates throughout the world (Ma et al., 1996; Khaleghi and Khadivi, 2020). Roses are one of the most important commercial plants, and as the queen of flowers are very important due to their usage in high value essential oil production and as garden rose, potted plants and cut flowers. The rose essential oil is used in perfumes for their sweet and long lasting fragrance (Muiruri et al., 2011). Rosa damascena is one of the most important spe- cies of Rosa genus, commonly known as Damask rose (known as Gole Mohammdi in Iran), which is cultivated in Bulgaria, France, Italy, Turkey, Iran, Morocco, USA, and India. R. damascena, a beautiful aromatic flower with immense horticultural importance, is one of the oldest and most valuable species. In addition, it has many appli- cations in the perfume, cosmetic, and food industries in- cluding production of rosewater, jam, jellies, conserves. This species is also used worldwide for manufacture of products with diverse applications such as aromathera- peutic, anti-HIV, antibacterial, antioxidant, antidepres- sant, antimicrobial, antiseptic, astringent agent, antispas- modic, sedative and blood cholesterol altering (Ozkan et al., 2004; Khaleghi and Khadivi, 2020). Miniature roses are a type of roses that are smaller in mass than the others. Miniature roses are character- ized with a prolonged flowering and could be used for creation of borders, flower beds, dwarf rose trees as well as a pot culture (Younis et al., 2015; Brailko et al., 2017). Roses are generally propagated by vegetative meth- ods like cutting, layering, budding and grafting. In ad- dition, seeds are used for propagation of species, new cultivars and production of rootstocks. Root suckers are the traditional and the most common rose propagation method, but this method is very low as it is seasonally de- pendent. Currently, in vitro micropropagation methods save time as well as can produce large numbers of plants within a small physical space (Pierik, 1991). Further, tis- sue culture permits manufacturing genetically similar and without disease plant material (Kadhimi et al., 2014; Cai et al., 2015). Micropropagation of rose species and their hybrids ranged from easy to difficult. Generally, plants with higher secondary metabolite contents are less suitable for growing in in vitro culture. It has been report- ed that a cytokinin such as 6- benzylamino purine (BAP) and an auxin, mostly 1-naphthalene acetic acid (NAA), or 2,4-dichlorophenoxyacetic acid (2,4-D) are normally included in the primary culture medium and have es- sential role on shoot proliferation in roses (Pati et al., 2010, Ahmadian et al., 2013). Indole-3 acetic acid (IAA) causes enlargement of plant cells, cell division, lateral branching of shoots and roots and vascular differentia- tion (Hobbie et al., 2000). Successful micro-propagation of some rose cultivars has been reported previously (Pati et al., 2010; Mahmoudi Noodezh et al., 2012). However, the success of these methods is dependent on the culti- var and genetic background of the plant. Some cultivars do not response to in vitro conditions; their proliferation and rooting rate is slow and many plantlets die during acclimatization (Alsemaan, 2013). Most studies on rose propagation have been carried out in Rosa damascena and only few studies on miniature rose. There are reports showing that rose micropropagation depends on cultivar genotype, the type and age of explant and type of culture media. The objective of the current study was to inves- Fig. 1A-C: Rose species. A: miniature rose, B, C: Damask rose Acta agriculturae Slovenica, 119/1 – 2023 3 Comparison of shoot and root regeneration of miniature potted rose (Rosa x hybrida L.) and Damask rose (R. damascena Mill.) ... tigate and compare the direct in vitro regeneration and proliferation of two rose species (Damask rose and min- iature rose). Further, the effect of different combinations of plant growth regulators and different strengths of MS media (full and half) in solid and liquid media were stud- ied and compared. 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL Explants are one of the primary factors for the effi- cient regeneration of in vitro plant cultures. In this study, nodal segments of Rosa damascene (Damask rose) and miniature rose (cultivar Modern Hybrid) were used as the explants for in vitro culture establishment (Fig. 1). Newly sprouted and actively growing young branches were collected from 3–4 years old stock plants growing in Lalehzar area in Keman province, Iran. Foliar parts were removed and branches were cut into segments with 1–2 nodes per segment. These nodal shoot segments were surface-sterilized by washing in detergent for 10 min. Then, they were kept under tap water for 30 min, dipped in 70  % ethanol for 1 min, immersed in 10  % sodium hypochlorite plus 1 % Tween 20 for 3 min, and embed- ded in with 0.1 % HgCl2 plus 1 % Tween 20 for 3 min, followed by 3 rinses with sterile distilled water. The last stage was embedding in antibiotics (100 mg l–1 ampicillin and tetracycline) for 20 min each (Tarrahi and Rezane- jad, 2013). 2.2 MS MEDIUM PREPARATION AND SHOOT INITIATION AND MULTIPLICATION The rate of tissue growth and morphogenetic re- sponses are highly affected by media features. The medi- um consisted of Murashige and Skoog (1962) basal salts and vitamins supplemented with different concentrations of growth regulators including different combinations of BAP, 2,4-D, NAA and gibberellic acid (GA3), sucrose (30 g l–1), and agar (8 g l–1) (16 different media, Tab. 1). The pH was adjusted to 5.7-5.8 before autoclaving. All media were sterilized by autoclaving for 20 min at 121 °C (1.5 kg cm–2 pressure). 15 sterilized explants were cultured on each petri dish containing autoclaved medium. Three petri dishes (3 repetitions) were used for each treatment and kept under a temperature of 25 ± 2°C and 16/8 h (light/dark) photoperiod. Light intensity of 23 μmol m-2 s-1 provided by cool white fluorescent tubes was used for shoot induction and proliferation and 11.5 μmol m-2 s-1 for root induction and growth. Explants were then rou- tinely subcultured onto medium of the same composi- tion in two weeks intervals. 2.3 ROOT INDUCTION AND GROWTH Root formation and growth of healthy shoots (1.5- 2 cm long) were studied and compared in both species using different strengths (full and half) of solid and liq- uid basal MS media supplemented with various concen- trations of IAA (24 media, Tab. 2). For the highest root formation frequency, before transferring 1.5-2 cm shoots into rooting media, two pretreatments were utilized: one set of shoots were floated in 500 mg l-1 IAA for 1 min, and other set were cultured on solid MS containing 3 mgl -1 2,4-D for 2 weeks. Thus, 24 various media were used for root initiation and growth (Tab. 2). 2.4 EXPERIMENTAL DESIGN AND STATISTICAL ANALYSIS The experiments were conducted as a completely randomized design. Data are expressed as mean ± stand- ard error (SE). Analysis of variance (ANOVA) was used Plant growth regulators (mg l-1) Media GA32, 4-DBAPNAA 0.1010.11 0.101.50.12 0.1020.13 0.102.50.14 0.1010.055 0.101.50.056 0.1020.057 0.102.50.058 0.10.1109 0.10.11.5010 0.10.12011 0.10.12.5012 0.10.051013 0.10.051.5014 0.10.052015 0.10.052.5016 Tab. 1: Different types of culture media used in shoot induc- tion and proliferation of Damask rose and miniature rose Each experiment had three replicates containing at least eight explants in each culture vessel Acta agriculturae Slovenica, 119/1 – 20234 F. REZANEJAD et al. to compare the means. Duncan’s test (p < 0.05) was employed to determine significant differences between means. Statistical analysis was conducted using the SPSS software. Each experiment had three replicates contain- ing minimum eight explants in each culture vessel. 3 RESULTS 3.1 SHOOT INDUCTION AND PROLIFERATION IN DIFFERENT CULTURE MEDIA The sterilized nodal explants were inoculated on different media containing different hormonal combina- tions and their shoot induction and proliferation were compared (Tabs. 3, 4, Figs. 2, 3). In miniature rose, the highest shoot proliferation (100 %) was obtained in the presence of 2 mg l-1 BAP and concentrations of 0.05 and 0.1 mg l-1 NAA (media 3 and 7) whereas in R. damascena, the highest levels of proliferation were observed in the presence of 2.5 mg l-1 BAP and 0.05 mg l-1 of 2, 4-D (me- dium 16) (Tab. 4). Also, shoot proliferation in medium 6 for miniature rose and media 4, 7, 8, 12 for R. dama- scena, was higher than 90  % (Tab. 4). In these optimal media (more than 90% proliferation), the axillary buds were sprouted earlier in miniature rose compared with R. damascena. In miniature rose, shoot induction and pro- liferation were observed about 5 and 17 days after plant- ing of single nodes respectively while these two phases occurred in R. damascena about 16 and 38 days after cul- turing respectively (Tab. 3 and Figs. 2, 3). 3.2 ROOT FORMATION AND GROWTH Root formation and growth of healthy shoots were studied on various combinations of IAA under different strengths of MS media (full and half) in solid and liquid Medium number Pretreatments Medium type Strength of MS medium IAA (mg l-1) 1 Floating explants in 500 mg l-1 IAA for 1 min as pretreatment Solid Full 0.10 2 Solid Full 0.05 3 Solid Full 0.00 4 Solid Half 0.10 5 Solid Half 0.05 6 Solid Half 0.00 7 Liquid Full 0.10 8 Liquid Full 0.05 9 Liquid Full 0.00 10 Liquid Half 0.10 11 Liquid Half 0.05 12 Liquid Half 0.00 13 Culturing explants on solid MS containing 3 mg l-1 2,4-D for two weeks as pretreatment Solid Full 0.10 14 Solid Full 0.05 15 Solid Full 0.00 15 Solid Half 0.10 17 Solid Half 0.05 18 Solid Half 0.00 19 Liquid Full 0.10 20 Liquid Full 0.05 21 Liquid Full 0.00 22 Liquid Half 0.10 23 Liquid Half 0.05 24 Liquid Half 0.00 Tab. 2: Different types of culture media used in rooting of Damask rose and miniature rose Each experiment had three replicates containing at least eight explants in each culture vessel Acta agriculturae Slovenica, 119/1 – 2023 5 Comparison of shoot and root regeneration of miniature potted rose (Rosa x hybrida L.) and Damask rose (R. damascena Mill.) ... culture media. Further, two pretreatments were utilized for best efficiency. In optimal media, root initiation and growth of miniature potted rose was observed earlier than R. damascena. In miniature rose, root initiation and growth were observed about 78 and 92 days after cultur- ing respectively while in R. damascena, root emergence and growth were recorded 125 and 138 days after trans- ferring to first medium for shoot induction respectively. Therefore, roots were initiated 42 and 67 days after trans- ferring to rooting media respectively in miniature rose and Damask rose (Tab. 3 and Figs. 2, 3). The results of rooting rate in various media revealed that the successful root formation just occurred in three media for miniature rose and two media for R. dama- scena. In both species, rooting frequency was higher in the half strength MS liquid medium than half strength MS solid medium (Tab. 5). In miniature rose, these three media are as follows: 1- the half strength MS liquid medium containing 0.05 mg l-1 IAA floated in 500 mg l-1 IAA (for one minute) as pretreatment with rooting frequency of 60 %. 2- half strength MS liquid medium without IAA, pretreatment in solid MS medium containing 3 mg l-1 2, 4-D for 2 weeks, with a rooting rate 62 %). 3- the half strength MS solid medium containing 0.05 mg l-1 IAA pretreated in solid MS medium containing 3 mgl-1 2, 4-D for 2 weeks, with root formation at a rate of 29% (Tab. 5, the under- lined values). Days Species Root length 2 cm Root Initiation Transfer to the rooting medium Shoot proliferation (1.5-2 cm long) Shoot induction 91.6777.6735.3315.675.33miniature rose 137.67124.6757.6737.6715.67Damask rose Tab. 3: The comparison of shoot and root regeneration of miniature rose and Damask rose in optimal medium Proliferation (%)Growth regulators (m/l) Media number Damask roseminiature roseGA3NAA2, 4-DBAP 57.78 ± 1.20cd 0.88d±57.78 0.100.10-1.001 62.22 ± 1.45cd 1.53abcd ±73.33 0.100.10-1.502 ± 1.53 cd66.67 0.34a0±1000.100.10-2.003 0.33a ±97.78 0.88d±57.78 0.100.10-2.504 2.20cd±62.22 0.88abc±88.89 0.100.05-1.005 1.15cd±60 0.57ab±93.33 0.100.05-1.506 0.67ab ±91.110.00a ±100 0.100.05-2.007 1.00ab ±93.33 1.20bcd±71.110.100.05-2.508 11.15cd ±60 0.88cd±62.22 0.10-0.101.009 1.20cd ±57.78 2.33bcd±71.110.10-0.101.5010 1.53cd ±53.33 1.33d ±55.56 0.10-0.102.0011 0.33ab ±95.56 0.53d ±53.33 0.10-0.102.5012 0.88bc ±75.56 0.57 d ±6 0.10-0.051.0013 1.00cd ±53.33 1.76cd ±64.44 0.10-0.051.5014 0.33d ±51.11 1.56abcd ±8 0.10-0.052.0015 0.41a ±100 1.86abcd±77.78 0.10-0.052.5016 Tab. 4: The comparison of the effects of different combinations of plant growth regulators on the proliferation of Damask rose and miniature rose Each experiment had three replicates containing at least eight explants in each culture vessel. Data are expressed as mean ± standard error, values with same letters in the same column are not significantly different (p ≤ 0.05) using Duncan’s multiple range test Acta agriculturae Slovenica, 119/1 – 20236 F. REZANEJAD et al. In R. damascena, rooting percentage obtained about 53 % in half strength MS liquid medium containing 0.05 mg l-1 IAA (pretreated onto solid MS medium contain- ing 3 mg l-1 2,4-D for 2 weeks). The other optimal me- dium with 32 % rooting percentage was recorded in half strength MS solid medium containing 0.1 mg l-1 IAA (floated in 500 mg l-1 IAA for 1 min as pretreatment) (Tab. 5, the underlined values). 4 DISCUSSION MS medium has been reported as the most com- mon basal medium used for rose micro-propagation. In addition, modified MS and ½MS media have been used successfully in various studies of rose species. Mahmoudi Noodezh et al. (2012) utilized a modified MS medium with higher levels of nitrates, calcium, and iron supple- mented with 4 mg l-1 6-benzylaminopurine and 0.25 mg l-1 indole-3-acetic acid for shoot initiation and prolifera- tion in R. damascena. Also, their results showed that a liquid half-strength medium supplemented with 1 mg l-1 IBA is the most successful medium for in vitro rooting in this cultivar. Badzian et al. (1991) reported that medium containing ½MS and 1g l-1 activated charcoal was ap- propriate for root formation in miniature rose cultivars (Badzian et al., 1991). In this study, the induction and growth of shoots and roots of two rose species were investigated in 16 different media for shoot initiation and growth and 24 different media for rooting. Proliferation is the most important stage of micropropagation and hence a successful pro- tocol with high efficiency is needed to increase its qual- ity. Cytokinins are the main growth regulators during proliferation. The induction and growth rate of explants depends upon many factors like season of sampling, age Fig. 2: A-I: Different stages of miniature rose propagation in optimal medium. A, B: The first stage of shoot induction and growth (swelling); C-F: Shoot proliferation and formation of the multi-leaf shoots (1.5-2 cm long); G-I: Transfer to the rooting medium and shoot and root growth (I, 78 days after initial culture) Acta agriculturae Slovenica, 119/1 – 2023 7 Comparison of shoot and root regeneration of miniature potted rose (Rosa x hybrida L.) and Damask rose (R. damascena Mill.) ... and portion of the branch, culture media, cultivar type, growth regulators, moisture and nutrient status (Pati et al., 2006). The concentrations of 2-2.5 mg l-l BAP, 0.1 mg l-1 GA3 and low levels of NAA, were appropriate for the highest proliferation rate in two studied species. The highest rate (100 %) of shoot proliferation was obtained in media 3 and 7 for miniature rose and medium 16 for R. damascena. The studies have been shown that concen- trations 1-10 mg l-1 BAP are required for bud break, pro- liferation and growth of shoots (Pati et al., 2006). Davoudi Pahnekolayi et al (2015) reported that the highest shoot proliferation in Rosa canina L. was ob- tained on Van der Salm (VS) medium containing 2 mg l⁻1 BAP compared with MS medium. Furthermore, the highest root induction obtained in ½ VS containing 0.6– 0.9 mg l⁻1 of NAA or IBA. BAP is necessary for prolif- eration, although the auxins particularly NAA, IAA and IBA in combination with BAP simultaneously improve the formation of the shoots. They indicated that NAA was more effective than other auxins (Davoudi Pahne- kolayi et al., 2015). Kim et al. (2003) reported the high- est rate of shoot proliferation in the presence of 2 mg l-1 BAP and 0.01 mg l-1 NAA in full-strength MS medium (Kim et al., 2003). Thi et al. (2008) demonstrated that the most suitable concentration for shoot initiation and mul- tiplication of roses was observed on MS medium supple- mented with 3 mg l-1 BAP (Thi et al., 2008). The highest number of shoots in rose ‘Morrasia’ was produced in 3 mg l-1 BAP (Asadi et al., 2009). In Rosa chinensis, differ- ent concentrations of BAP (0, 0.5, 1, 1.5, 2 mg l-1) (BA) and Thidiazuron (1.5 mg l-1)) induced shoot production with a percentage of 100 % (Tibkwang et al., 2018). Quick Fig.3: A-J: Different stages of R. damacesna propagation in optimal medium. A, B: Initial stage of shoot induction (swelling); C, F: The stage of appearance and initial growth of shoots; E-F: Proliferation or the multi-leaf stage of shoots (1.5-2 cm long), G-J: Transfer to the rooting medium and root growth (J, 125 days after initial culture) Acta agriculturae Slovenica, 119/1 – 20238 F. REZANEJAD et al. deep treatment of microshoots in auxin compounds have been reported frequently (Kumar et al., 2000; Nikbakht et al., 2005). In this study, the highest proliferation rate of shoots in studied species was obtained in higher concen- trations of BAP and lower amount of auxins and GA3. In optimal medium, the axillary buds were sprouted earlier in miniature rose than R. damascena. Cultivar, explant type and medium composition are considered as three main factors affecting in vitro plant regeneration in many plant species (Gubis et al., 2003, Bidabadi and Jain, 2020). In present study, the significant differences were ob- served in regeneration capacity between two species as well as between different combinations of culture media. Root induction is affected by different external and inter- nal factors, among them the height and age of shoots are important factors (Pati et al., 2006). Exogenous auxins were shown to increase the availability of carbohydrates at the site of root development (Abidin and Metali, 2015). According to a study by Jabbarzadeh and Khosh-Khui (2005) in roses, the best treatment for rooting of shoots was 2.5 mg l-1 of 2,4-D for 2 weeks in MS medium (as pretreatment) and then transferring the explants to hor- mone free MS medium (Jabbarzadeh and Khosh-Khui, 2005). In current study, the 2, 4-D induced root induc- tion successfully. It has been reported that 2, 4-D prevent the failure and degradation of the endogenous auxins through the oxidase enzymes and lead to root induction (Jabbarzadeh and Khosh-Khui, 2005). Root induction with 2,4 -D has also been reported in roses and other plants (Edwin and Paul, 1984). Although rooting occurs in both solid and liquid media but there are significant differences in the rooting potential of the two media. In the two species studied, rooting frequency was higher in the half strength MS media than in the full strength media. Moreover, root formation and growth was higher in MS liquid medium than solid one. The highest root- ing frequencies were 62  % and 53  % in miniature rose and Damask rose respectively. Similarly, Pati et al. (2006) reported the highest percentage of rooting in liquid me- dium (85 %) compared with solid media (5 %) suggesting that low osmotic potential in solid medium reduces the root induction (Nikbakht et al., 2005; Pati et al., 2006). According to the results, similar to shoot induction and proliferation, rooting in miniature rose was faster than in R. damascena. A lower rooting ability was also cited in old garden roses (R. damascena and R. canina) compared with modern (new) ones (R. hybrid) (Pati et al., 2006). Kirichenko et al. (1991) reported that micro shoots of the essential oil bearing roses have higher rooting problems and are rooted worse than the ornamental and modern varieties (Kirichenko et al., 1991). Nikbakht et al. (2005) reported that in vitro rooting of old roses including Damask rose is much more difficult than modern roses (Nikbakht et al., 2005). Similarly, in current study, R. damascena as an old species with the highest potential essential oils revealed the lower percentage of rooting Species Pretreatments PGR Root induction (%) IAA Solid MS medium Liquid MS medium Full strength Half strength Full strength Half strength Miniature rose Dipping (floating) in 500 mg l -1 IAA 0 - - - - 0.05 - - - 1.5a ±60 0.1 - - - - culturing on solid MS containing 3 mg l -12,4-D 0 - - - a1.2±62.2 0.05 - 28.8 ± 0.67b - - 0.1 - - - - Damask rose Dipping (floating) in 500 mg l -1 IAA 0 - - - - 0.05 - - - - 0.1 - 0.9b ±31.1 - - culturing on solid MS containing 3 mg l- 12,4-D 0 - - - - 0.05 - - - 1.8a± 53.3 0.1 - - - - Tab. 5: The rooting rate (%) of miniature rose and Damask rose under two pretreatments, different combinations of IAA (0, 0.1, 0.05 mgl-1) and different strengths of MS media (full and half) in solid and liquid media (24 various media) Each experiment had three replicates containing at least eight explants in each culture vessel. Data are expressed as mean ± standard error, values with the same letters in the same column are not significantly different (p ≤ 0.05) using Duncan’s multiple range test Acta agriculturae Slovenica, 119/1 – 2023 9 Comparison of shoot and root regeneration of miniature potted rose (Rosa x hybrida L.) and Damask rose (R. damascena Mill.) ... compared with miniature rose. Further, it has been re- ported that there are genes that are involved in shoot and root formation and growth. Also, the possible involve- ment of the gene in modulating hormone levels has also been reported (Ginova, 2012). In conclusion, in vitro culture methods of roses are important procedures in production of new and adapt- able cultivars, eliminating incompatible rootstocks and fast formation of superior cultivars and rootstocks. In present study, the significant differences were observed in regeneration capacity between two species as well as between different combinations of culture media. The results showed that MS medium supplemented with low concentrations of plant growth regulators were re- sulted in 100 % shoot proliferation in both species. The 2, 4-D induced root induction successfully. The root- ing frequency was higher in the half strength MS liquid medium than the others. The highest rooting frequency was 62  % and 53  % respectively in miniature rose and Damask rose. The shoot and root formation were faster and higher in miniature potted rose compared with R. damascena as an old species with highest potential es- sential oils. 5 ACKNOWLEDGEMENTS This work was financially supported by Shahid Ba- honar University of Kerman, Iran. The authors acknowl- edge financial support from the University of Kerman. 6 REFERENCES: Abidin, N., and Metali, F. (2015). Effects of different types and concentrations of auxins on juvenile stem cuttings for prop- agation of potential medicinal Dillenia suffruticosa martelli shrub. Research Journal of Botany, 10(3), 73-87. https://doi. org/10.3923/rjb.2015.73.87 Ahmadian, E., Lolaei, A., Mobasheri, S., Bemana, R. (2013). Investigation of importance parameters of plant tissue (review). International Journal of Agriculture and Crop Sci- ences, 5(8), 900-905. Alsemaan, T. (2013). Micro-propagation of Damask Rose (Rosa damascena Mill.) cv. Almarah. International Journal of Ag- ricultural Research, 8(4), 172-177. https://doi.org/10.3923/ ijar.2013.172.177 Asadi, A.A., Vedadi, C., Rahimi, M., Naserian, B. (2009). Effect of plant growth hormones on root and shoot regeneration in rose (Morrasia) under in-vitro conditions. Bioscience Re- search, 6(1), 40-45. Badzian, T., Hennen, G.R., Fotyma-Kern, J. (1991). In vitro rooting of clonal propagated miniature rose cultivars. In International Symposium on Plant Biotechnology and its Contribution to Plant Development, Multiplication and Improvement, 289, 329-330. https://doi.org/10.17660/Acta- Hortic.1991.289.81 Bidabadi, S.S., Jain, S. M. (2020). Cellular, molecular, and physi- ological aspects of in vitro plant regeneration. Plants, 9(6), 702. https://doi.org/10.3390/plants9060702 Brailko, V.A., Plugatar, S.A., Pilipchuk, T.I., Plugatar, Y.V., Mitrofanova, I.V. (2017). Morphological and physiological features of the miniature rose cultivar Risen Shine under long time culture in vitro and in vivo. In VII International Symposium on Production and Establishment of Micropro- pagated Plants, 1224, 139-144. https://doi.org/10.17660/ ActaHortic.2018.1224.19 Cai, Z., Jing, X., Tian, X., Jiang, J., Liu, F., Wang, X. (2015). Di- rect and indirect in vitro plant regeneration and the effect of brassinolide on callus differentiation of Populus euphratica. South African Journal of Botany, 97, 143-148. https://doi. org/10.1016/j.sajb.2015.01.006 Davoudi Pahnekolayi, M., Samiei, L., Tehranifar, A., Shoor, M. (2015). The effect of medium and plant growth regulators on micropropagation of Dog rose (Rosa canina L.). Journal of Plant Molecular Breeding, 3(1), 61-71. Edwin, F.G., Paul, D.Sh. (1984). Plant Propagation by Tissue Culture. Handbook and Directory of Comerical Laborato- ries. Exegetics Ltd., Eversley, Basingstoke, Hants, UK. Ginova, A., Tsvetkov, I., kondakova, V. (2012). Rosa damascene Mill. An overview for evaluation of propagation methods. Bulgarian Journal of Agricultural Science, 18, 545-556. Gubis, J., Lajchova, Z., Farago, J. Jurekova, Z. (2003). Effect of genotype and explant type on shoot regeneration in to- mato (Lycopersicon esculentum) in vitro. Czech Journal of Genetics and Plant Breeding, 39(1), 9-14. https://doi. org/10.17221/3715-CJGPB Hobbie, L., McGovern, M., Hurwitz, L.R., Pierro, A., Liu, N.Y., Bandyopadhyay, A., Estelle, M. (2000). The axr6 mutants of Arabidopsis thaliana define a gene involved in auxin re- sponse and early development. Development, 127(1), 23-32. https://doi.org/10.1242/dev.127.1.23 Jabbarzadeh, Z., Khosh-Khui, M. (2005). Factors affecting tis- sue culture of Damask rose (Rosa damascena Mill.). Scientia Horticulturae, 105(4), 475-482. https://doi.org/10.1016/j. scienta.2005.02.014 Kadhimi, A.A., Alhasnawi, A.N., Mohamad, A., Yusoff, W.M.W., Zain, C. (2014). Tissue culture and some of the factors affecting them and the micropropagation of straw- berry. Life Science Journal, 11(8), 484-493. Khaleghi, A., Khadivi, A. (2020). Morphological characteri- zation of Damask rose (Rosa× damascena) germplasm to select superior accessions. Genetic Resources and Crop Evo- lution, 67(8), 1981-1997. https://doi.org/10.1007/s10722- 020-00954-z Khaskheli, A.J., Khaskheli, M.I., Khaskheli, M.A., Shar, T., Ahmad, W., Lighari, U.A., Khaskheli, M.A., Khaskheli, A.A. and Makan, F.H. (2018). Proliferation, multiplica- tion and improvement of micro-propagation system for mass clonal production of rose through shoot tip culture. American Journal of Plant Sciences, 9, 296-310. https://doi. org/10.4236/ajps.2018.92024 Kim C.K., Oh J.Y., Jee S.O., Chung J.D. (2003). In vitro micro- Acta agriculturae Slovenica, 119/1 – 202310 F. REZANEJAD et al. propagation of Rosa hybrid L. Plant Biotechnology, 5, 115- 119. Kirichenko, E.B., Kuz-Mina, T.A., Kataeva, N.V. (1991). Factors in optimizing the multiplication of ornamental and essen- tial oil roses in vitro. Byulleten-Glavnogo-Botanicheskogo Sada, 159, 61–67. Ma, Y., Byrne, D. H., Chen, J. (1996). Propagation of rose spe- cies in vitro. In vitro Cellular and Developmental Biology - Plant, 32(2), 103-108. https://doi.org/10.1007/BF02823139 Mahmoudi Noodezh H.M., Moieni, A., Baghizadeh, A. (2012). In vitro propagation of the Damask rose (Rosa damascena Mill.), In Vitro Cellular and Developmental Biology – Plant, 48(5), 530-538. https://doi.org/10.1007/s11627-012-9454-z Muiruri, S.N., Mweu, C.M. and Nyende, B.A. (2011). Micro- propagation protocols using nodal explants of selected rose (Rosa hybrida) cultivars. African Journal of Horticultural Science, 4, 60-65. Murashige, T., Skoog, F. (1962). A revised medium for rap- id growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473-497. https://doi. org/10.1111/j.1399-3054.1962.tb08052.x Nikbakht, A., Kafi, M., Mirmasoudi, M., Babalar, M. (2005). Micropropagation of damask rose (Rosa damascena Mill.) cvs Azaran and Ghamsar. International Journal of Agricul- ture and Biology, 7(4), 535-538. Ozkan, G., Sagdic, O., Baydar, N.G., Baydar, H. (2004). Note: Antioxidant and antibacterial activities of Rosa damascena flower extracts. Food Science and Technology International, 10(4), 277-281. https://doi.org/10.1177/1082013204045882 Pati, P.K., Rath, S.P., Sharma, M., Sood, A., Ahuja, P.S. (2006). In vitro propagation of rose- a review. Biotechnol- ogy advances, 24(1), 94-114. https://doi.org/10.1016/j.bio- techadv.2005.07.001 Pati, P.K., Kaur, N., Sharma, M. and Ahuja, P.S. (2010). In vitro propagation of rose. In Protocols for in vitro Propagation of Ornamental Plants, 163-176. https://doi.org/10.1007/978- 1-60327-114-1_16 Pierik, R.L.M. (1991). Horticulture new technologies and ap- plications proceeding of the international seminar on new frontiers in horticulture. Current Plant Science and Biotechnology in Agriculture, 12, 141–153. https://doi. org/10.1007/978-94-011-3176-6_23 Tarrahi, R., Rezanejad, F. (2013). Callogenesis and production of anthocyanin and chlorophyll in callus cultures of veg- etative and floral explants in Rosa gallica and R. hybrida (Rosaceae). Turkish Journal of Botany, 37(6), 1145-1154. https://doi.org/10.3906/bot-1205-42 Thi, K.O., Khai, A.A., Lwin, K.M. (2008). Micropropagation of Rose (Rosa hubrida spp.) by in vitro culture technique. GMSARN International Conference on Sustainable Develop- ment: Issues and Prospects forthe GMS, 12-14. Tibkwang, A., Junkasiraporn, S., Chotikadachanarong, K. (2018). Effects of Cytokinnin and Sucrose on Tissue Cul- ture of Rosa chinensis. Burapha Science Journal, 23(2), 712- 721. Younis, A., Riaz, A., Javaid, F., Ahsan, M., Tariq, U., Aslam, S. Majeed, N. (2015). Influence of various growing substrates on growth and flowering of potted miniature rose cultivar “Baby Boomer”. Specialty Journal of Agricultural Sciences, 1(2), 28-33.