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Article

Influence of Cytokinins, Dark Incubation and Air-Lift Bioreactor Culture on Axillary Shoot Proliferation of Al-Taif Rose (Rosa damascena trigintipetala (Diek) R. Keller)

by
Ali Mohsen Al-Ali
,
Yaser Hassan Dewir
* and
Rashid Sultan Al-Obeed
Plant Production Department, College of Food and Agricultural Sciences, King Saud University, Riyadh 11451, Saudi Arabia
*
Author to whom correspondence should be addressed.
Horticulturae 2023, 9(10), 1109; https://doi.org/10.3390/horticulturae9101109
Submission received: 12 September 2023 / Revised: 3 October 2023 / Accepted: 5 October 2023 / Published: 7 October 2023
(This article belongs to the Special Issue Plant Biotechnology: Applications in In Vitro Plant Conservation and Micropropagation)
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Abstract

:
Rose is a widely favored floriculture crop that is commercially propagated through the application of tissue culture techniques. Here, we report an effective method for axillary shoot proliferation in Al-Taif rose, an important cultivar for rose oil industry. Stem nodes were excised from an adult donor Al-Taif rose shrub and cultured for 4 weeks on Murashige and Skoog’s (MS) medium supplemented with 6-benzylaminopurine (BAP) or gibberellic acid (GA3) at 0 and 3 mg·L−1 to induce the sprouting of axillary shoots. Al-Taif rose shoots were cultured in vitro for 6 weeks on MS medium fortified with different concentrations of cytokinins, light/dark incubation and different culture types (gelled and liquid/bioreactor culture). The culture conditions that were applied had a noteworthy impact on the responses of Al-Taif rose shoot proliferation. The supplementation of the medium with 6-benzylaminopurine (BAP) resulted in an augmented rate of shoot proliferation in comparison to other cytokinins. Additionally, dark incubation limited foliage growth, leaf yellowing and abscission and favored shoot proliferation compared with light incubation. Liquid culture using bioreactors provided higher axillary shoot proliferation and growth as compared with gelled culture. A continuous immersion system with a net provided the highest axillary shoots (four shoots per explant) and shoot length (16.5 cm), whereas an immersion system without a net provided the highest fresh weight of axillary shoots (499 mg per explant). These findings will improve commercial propagation and contribute to the rose oil industry of Al-Taif rose.

1. Introduction

The genus Rosa is a member of the family Rosaceae and comprised of 366 accepted plant species [http://www.theplantlist.org/1.1/browse/A/Rosaceae/Rosa/ accessed on: 9 September 2023]. This genus comprises numerous plant species of significant economic value, exemplified by the Damask rose (Rosa damascena Herrm). This particular species is extensively utilized on a global scale for the production of various commodities, spanning from aroma-therapeutics and astringents to sedatives and blood cholesterol-altering products. Furthermore, it possesses notable antibacterial, antiseptic, antidepressant, antimicrobial [1], antioxidizing [2] and anti-HIV properties [3]. Moreover, it possesses numerous utilizations in the realm of food enterprises, encompassing the creation of rosewater and desiccated blossoms. The employment of rose petals is employed in the making of marmalades, preserves and jellies. The remnants from the process of distillation can be employed for the nourishment of livestock and the practice of composting [4]. The Damask rose is also utilized as a decorative flora in parks and gardens. Rosa damascena was originally introduced from the Middle East into Western Europe. The rose of Al-Taif (Rosa damascena trigintipetala (Diek) R. Keller) is an infraspecific taxon of Rosa damascena [http://www.theplantlist.org/1.1/browse/A/Rosaceae/Rosa/ accessed on: 9 September 2023]. This variety of Damask rose is considered one of best rose species cultivated for rose oil production [5,6] and well known for its deep and intensive fragrance in the Arabian region. This oil-rich 30-petal rose has been cultivated in Al-Taif for three centuries. Al-Taif city is characterized by its favorable temperatures (the western part of Al-Taif city rises over 2000 m above sea level), plentiful groundwater, well-established irrigation systems and fine top soil. These advantages are combined to earn the name “Arabia’s Rose”. The Al-Taif rose is considered one of the most valuable and ancient varieties. This crop has a significant economic role due to its substantial value in terms of exporting its oil to Arab countries, its decorative purposes, its medicinal applications and its utilization in the manufacture of perfumes [7].
There are several methods for conventional vegetative propagation of rose. The propagation methods commonly employed for the Damask rose include cuttings and suckers. Nonetheless, these approaches are time-consuming, slow and often associated with various problems such as the restricted availability of stock plants and an extended timeframe for production [8]. In addition, certain techniques are applicable to certain varieties of roses while being unsuitable for others. Suitable rootstock for the production of grafted material from oil-bearing roses has been established in order to accelerate the reproduction of mother crops. It was found that Rosa canina Brogs is the most suitable rootstock due to its high compatibility with Rosa damascena trigintipetala [9]. Propagation by layers requires a rather large area, and weed control among the layers is problematic. Knowledge of the strengths and weaknesses of the selected vegetative propagation technique is imperative for its effective application. [4]. Similarly, these methods are insufficient to support the increasing demand for healthy seedlings in Saudi Arabia.
Plant tissue culture techniques enable the rapid production of rose cultivars with desirable traits and facilitate the production of robust and pathogen-free plants [10]. The development of an efficient micropropagation procedure through axillary shoot multiplication is promising for the commercial production of high-quality Al-Taif rose plants. Micropropagation via axillary or shoot meristem proliferation in gelled cultures is regarded as a labor-intensive method of producing elite clones. The application of liquid cultures using bioreactors for in vitro propagation provide a workable means for the large-scale production of several plant species. The use of bioreactors could potentially resolve manual handling and decrease the production cost compared with gelled cultures. However, inconsistencies in optimizing the type of bioreactors and culture conditions have been reported [11]. The success of in vitro rose cultures is cultivar-dependent due to the genetic background of the plant. Bhattacharjee [12] emphasized the connection between plant propagation, soil characteristics and prevailing climate conditions. Some cultivars do not respond to the in vitro conditions or their proliferation rate is slow [13]. In this context, Rosa damascena is defined as a recalcitrant species. In the present study, the shoot proliferation and growth of Al-Taif roses were investigated in response to the type and concentrations of cytokinins, light and dark incubation and air-lift bioreactor culture.

2. Materials and Methods

2.1. Plant Material, Surface Disinfection of Explants and Bud Sprouting of Al-Taif Rose

This research was carried out at the laboratory of plant tissue culture, which is located in the College of Food and Agricultural Sciences at King Saud University. Semi-hard woody cuttings, 5–10 cm long, were taken from a two-year-old adult Al-Taif rose shrub (Rosa damascena trigintipetala (Diek) R. Keller) (Figure 1a). Explants were defoliated, followed by thorough rinsing under a continuous flow of tap water. Subsequently, the explants were sectioned into segments that contain a single node (Figure 1b,c). The explants were subsequently immersed in a solution rich in antioxidants (ascorbic acid at a concentration of 150 mg·L−1 and citric acid at a concentration of 100 mg·L−1) for a duration of 10 min. Following this step, the explants underwent a triple rinse using distilled water. Subsequently, they were subjected to a disinfection process for a period of 30 s in 70% ethanol. This was followed by an additional 15 min immersion in a solution containing 0.1% (v/v) mercuric chloride supplemented with 2–3 drops of Tween 20 (polyoxyethylene-sorbitan monolaurate). After rinsing three times with sterile distilled water, explants were cultured for 2 weeks on MS [14] medium containing 3% (w/v) sucrose and 3.0 mg·L−1 gibberellic acid (GA3) or 6-benzylaminopurine (BAP) for the sprouting of axillary shoots; MS medium without plant growth regulators served as a control. Prior to autoclaving at 121 °C for 20 min, the pH of all the medium variants was adjusted to 5.7. Additionally, the medium was gelled with 0.7% (w/v) Sigma agar-agar. All the cultures were incubated at 23 ± 2 °C for 2 weeks under dark conditions (to avoid browning of plant tissue) followed by 2 weeks under a 16 h photoperiod provided using cool white fluorescent lights. The intensity of the lights was set at 35 μmol m−2 s−1 photosynthetic photon flux (PPF). The contamination (%), bud break (%), number of days to bud break and length of sprouting bud were recorded.

2.2. Effects of Type and Concentrations of Cytokinins on Axillary Shoot Proliferation of Al-Taif Rose

Al-Taif rose axillary shoots (9 per culture vessel) were transferred to 100 mL MS medium supplemented with different concentrations of 6-benzylaminopurine (BAP; 0, 0.5, 1 and 2 mg·L−1), kinetin (0, 1, 3 and 5 mg·L−1), 2-isopentenyl adenine (2ip; 0, 1, 3 and 5 mg·L−1) and thidiazuron (TDZ; 0, 0.01, 0.1 and 0.5 mg·L−1). The cultures were incubated under a 16 h photoperiod provided by cool white fluorescent lights at 35 μmol m−2 s−1 photosynthetic photon flux (PPF). The shoot growth and shoot multiplication responses were assessed following a 6-week culture period, whereby the number of axillary shoots developed per explant and the shoot length as well as fresh weight of the whole explants were recorded. All measurements were obtained from 15 randomly selected explants.

2.3. Effects of Dark Incubation on Axillary Shoot Proliferation of Al-Taif Rose

In this experiment, axillary shoots of Al-Taif rose were cultured onto MS medium supplemented with various concentrations of 6-benzylaminopurine (BAP; 0, 0.5, 1 and 2 mg·L−1). The cultures were then subjected to either a 16 h photoperiod or complete darkness conditions. The remaining culture conditions were maintained as previously mentioned. The shoot growth and shoot multiplication responses were assessed after 6 weeks of culture. The assessment included the number of axillary shoots developed per explant and the shoot length as well as fresh weight of the whole explants. All measurements were obtained from 15 randomly selected explants.

2.4. Effects of Gelled/Bioreactor Culture on Axillary Shoot Proliferation of Al-Taif Rose

Continuous immersion bioreactor culture (with a net or without a net) was employed in order to evaluate and select an appropriate technique for the proliferation of axillary shoots of Al-Taif rose in liquid media. This method was then compared to gelled culture. A total of 80 explants per bioreactor were transferred to a balloon-type bubble air-lift bioreactor with a capacity of 3 L. This bioreactor, manufactured by PLT Scientific SDN BHD located in Puchong, Selangor D.E., Malaysia, was filled with 1200 mL of MS liquid medium. The medium was supplemented with 30 g·L−1 of sucrose and 0.5 mg·L−1 of BAP. Before autoclaving at a temperature of 121 °C and a pressure of 1.2 kg cm−2 for a duration of 20 min, the pH of the medium was adjusted to 5.8. In the immersion-type bioreactor, a plastic net was employed to facilitate the partial immersion of the explants in order to prevent their complete submersion in the liquid medium. The air input volume was adjusted to 0.1 vvm (air volume to culture volume per minute). The bioreactors and culture vessels were all maintained at a temperature of 25 °C ± 2 °C in conditions of darkness. Following a 6-week culture period, data pertaining to the number of proliferated shoots, fresh weight and shoot length were collected from a sample of 15 randomly selected explants.

2.5. Experimental Design and Data Analysis

All experiments were conducted in a completely randomized design. The impact of the treatments was evaluated using ANOVA and Tukey’s multiple range test in SAS (version 9.4; SAS Institute, Inc., Cary, NC, USA).

3. Results and Discussion

3.1. Establishment of Al-Taif Rose Aseptic Culture

Oxidative browning was encountered during the culture initiation of Al-Taif rose, using nodal explants that were incubated under light conditions (Figure 1d,e). Soaking nodal explants in an antioxidant solution, the inclusion of BAP or GA3 to the culture initiation medium and dark incubation prevented tissue browning (Figure 1f; Table 1). Nodal explants cultured onto medium containing 3 mg·L−1 of GA3 or BAP resulted in 100% bud break within 5.3–5.4 days and had the highest shoot length (5.8 and 4.8 cm, respectively). A low percentage of bud break (60%) and low values of shoot length and fresh weight were obtained on medium lacking plant growth regulators. Tissue browning is a result of the accumulation and subsequent oxidation of phenolic compounds in the tissue and in the culture medium [15,16]. Browning results in slow growth, low regeneration and ultimately leads to cell/tissue or plant death [15,17]. It has been documented to impede the successful establishment of cultures in various woody plant species such as the American phoenix tree (Platanus occidentalis) [18] and strawberry tree (Arbutus unedo) [19]. Several researchers employed antioxidant solutions to reduce or prevent browning in plant tissue cultures, e.g., button mangrove (Conocarpus erectus) [20], oak (Quercus robur) [21], Platanus occidentalis [18] and North American ginseng (Panax quinquefolius) [16]. Reduced tissue browning was linked to the inclusion of additives, such as PVP (polyvinyl pyrrolidone), citric acid or ascorbic acid, or through employing the strategy of regular subculturing [22] or incubating cultures in complete darkness for a duration of one to two days after inoculation. This was due to the fact that exposure to light was found to stimulate the activity of the enzyme polyphenol oxidase [23].
Dark incubation has been reported to influence shoot regeneration in vitro. Incubation in the dark may delay the degradation of endogenous and/or exogenous plant growth regulators and reduce cell wall thickness, thus facilitating the translocation of plant growth regulators [24] and in turn regulating the morphogenic responses. Ibrahim and Debergh [25] indicated that dark incubation for one week was optimal for shoot regeneration in rose (Rosa hybrida). Choi et al. [26] indicated that a duration of 4–5 weeks of dark incubation was essential for the shoot regeneration of Diospyros kaki cultivars ‘Nishimurawase’ and ‘Fuyu’. The sprouting of ginger (Zingiber officinales) buds was developed in MS medium under dark conditions for one month [27]. A minimum of one week of dark incubation was necessary in order to promote organogenesis in leaf explants of strawberry (Fragaria × ananassa) [28]. Similarly, Mitić et al. [29] indicated that pre-incubation in the dark proved to be an essential factor for the in vitro regeneration of apple (Malus domestica Borkh.). A positive effect of dark pretreatment on shoot proliferation was reported for ‘Marion’ Blackberry (Rubus sp) [30]. Mendi et al. [31] studied snake melon (Cucumis melo var. flexousus), and dark pretreatment for 7 days resulted the highest frequency of adventitious shoot regeneration (42.8%) recorded on MS medium fortified with 1.0 mg·L−1 BAP+ 0.25 mg·L−1 indole-3-acetic acid (IAA). These findings emphasize the essential requirements of dark incubation for in vitro morphogenetic response and that dark requirements are species- and genotype-specific.
An early study by Rout et al. [32] highlighted the importance of the cytokinin BAP for in vitro bud break in Rosa hybrida and Rosa multiflora. In medium containing BAP, the time required for bud break was about 4 and 6 days for Rosa multiflora and Rosa hybrida, respectively. Conversely, 16 days were required for bud break in media devoid of growth regulators. Similarly, the incorporation of BAP at concentrations ranging from 1.0 to 10.0 mg·L−1 in the growth medium played a crucial role in facilitating the initiation of bud growth and subsequent shoot multiplication in R. hybrida [33,34]. Our results on Al-Taif rose also confirm these previous findings. Additionally, GA3 proved effective for preventing browning and enhancing 100% bud break and shoot proliferation in Al-Taif rose. The proliferating shoots were healthier compared to shoots on medium devoid of GA3. GA3 has been documented as a regulator of the expression of the gene responsible for phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and as an inhibitor of the activity of the PAL enzyme; therefore, the inclusion of GA3 in the medium was found to overcome browning in tissue culture [20,35].

3.2. Axillary Shoot Proliferation of Al-Taif Rose in Response to Cytokinins

The proliferation of axillary shoots, the length of shoots and the fresh weight of Al-Taif rose explants were significantly affected by the type of cytokinins rather than the concentrations of cytokinins (Table 2; Figure 2a,b). The interaction effect of cytokinin type and concentration had significant effect on shoot length but did not affect the number of shoots and shoot fresh weight. Supplementation of the medium with BAP induced the highest number of axillary shoots (1.9 shoots per explant) compared to the control and other cytokinin treatments. Kinetin, 2ip and TDZ treatments did not stimulate shoot proliferation in Al-Taif rose as compared to the control. Kinetin and TDZ had the lowest number of shoots, smallest shoot lengths and lowest fresh weight per explant. In general, BAP had a greater effect than other cytokinins for shoot proliferation whereas 2ip and the control treatments resulted in the greatest shoot lengths (Table 2).
Previous studies highlighted the necessity of BAP for the in vitro shoot proliferation and development of rose plant species. Bressan et al. [36] reported that low BAP concentrations (0.03–0.3 mg·L−1) had promotive effects on the axillary shoot development of Rosa hybrida ‘Gold Glow’. A combined treatment of BAP at concentrations of 2.5–3 mg·L−1 with a low rate of indole-3-butyric acid (IBA; 0.1 mg·L−1) was the most suitable treatment for the in vitro multiplication of Rosa damascena (3.75–4.0 shoots per single-node explant) [37]. A BAP concentration of 1–2 mg·L−1 was found to be the most appropriate concentration for the in vitro propagation of Rosa damascena. [38,39]. Similarly, Carelli and Etcheverrigaray [40] compared the effects of three cytokinins applied at 3 mg·L−1 on the in vitro establishment of Rosa hybrida ‘Baronesse’ micropropagation and found that BAP produced more shoots (2.57 shoots) in comparison to 2iP (2.17) and Kinetin (1.73). Mamaghani et al. [41] investigated the impact of culture medium and a combination of various plant growth regulators on the shoot proliferation in three elite Iranian Rosa damascena accessions: M6 (Kashan), G1 (East Azerbyjan) and G2 (West Azerbyjan). The most significant shoot proliferation (5.9) was achieved when using a combination of 5 mg·L−1 BAP and 0.1 mg·L−1 thidiazuron (TDZ). The maximum shoot length was observed in the medium containing 0.5 mg·L−1 IAA, 5 mg·L−1 BAP and 0.01 mg·L−1 TDZ. Shoot multiplication and shoot length varied with genotype and BAP as well as kinetin concentration. M6 accession required 2 mg·L−1 BAP and 2 mg·L−1 Kinetin for maximum shoot multiplication and shoot length, while G1 and G2 required 2.5 mg·L−1 BAP and 2.5 mg·L−1 Kin. In general, BAP has been characterized as more efficacious for the multiplication of axillary shoots in various Rosa genotypes compared to 2-iP, Kinetin and TDZ [40,41,42,43,44]. The optimal concentration of BAP ranged from 0.5 to 5 mg⋅L−1, depending on the rose species and cultivar, as well as the physiological condition of the explants [45]. Variations in the activity of different cytokinins may be elucidated according to their distinct uptake rate observed in various genomes [46]. Moreover, 2iP possesses a double bond in its chemical structure, rendering it susceptible to the influence of cytokinin oxidases [47]. Elevated levels of cytokinins have the potential to enhance shoot proliferation and growth. Nevertheless, it is of utmost significance to simultaneously prevent the occurrence of abnormalities, such as the formation of calluses at the explant base or the formation of stunted shoots [48].
The efficacy of BAP, in comparison to other cytokinins, in stimulating shoot proliferation in vitro, has been substantiated in various woody plant species, including cheesewood (Pittosporum napaulensis) [49], cancer bush (Lessertia frutescens) [50] and in other members of the family Rosaceae such as Rosa centifolia [51]. TDZ is a potent synthetic cytokinin and has been proven to be effective for shoot organogenesis and morphogenesis in several plant species including woody plants [52,53]. However, in the present study, TDZ was not effective for shoot proliferation in Al-Taif rose; however, no morphological abnormalities were observed. TDZ has species-specific effects, and its ineffectiveness for shoot proliferation has been reported in plant species such as peace lily (Spathiphyllum cannifolium) [54] and European ash (Fraxinus excelsior) [55].
Previous studies highlighted that combined treatment with BAP and auxins could have a synergistic effect that increases shoot proliferation in rose species, e.g., Rosa hybrida [40] and Rosa damascena [37], as well as woody plant species, including the arjun tree (Terminalia arjuna) [56] and strawberry tree (Arbutus unedo) [19]. However, several authors reported the in vitro response of roses is genotype-dependent [57,58,59,60,61]. In our study, it should be noted that various BAP and IBA or naphthalene acetic acid (NAA) combinations had no positive effects on the axillary shoots of Al-Taif rose “data not presented”. The addition of auxins resulted in low proliferation and callus formation at the base of Al-Taif rose microshoots incubated either under light or dark conditions (Figure 3c,d, respectively). The control of shoot morphogenesis in vitro requires culture conditions which will limit leaf development and at the same time enhance bud proliferation [62]. In the present study, the foliage growth of Al-Taif rose was limited under dark conditions. This foliage limitation has several advantages towards the mass propagation of rose. It prevented leaf yellowing and abscission and facilitated the ease of sub-culturing and inoculating large number of shoots in the culture vessels (Figure 4).

3.3. Axillary Shoot Proliferation of Al-Taif Rose in Response to Gelled/Liquid Bioreactor Cultures

Bioreactor culture enhanced axillary shoot proliferation and growth compared with gelled culture (Figure 5 and Figure 6). The highest number of shoots (four per explant) was obtained with continuous immersion with a net, while continuous immersion without a net produced the greatest fresh weight per explant (499 mg). Gelled culture produced the lowest values of shoot length (6 cm), number of shoots (2.5) and fresh weight (84 mg). In the present study, the improved development of Al-Taif rose axillary shoots in a bioreactor culture system could be due to the higher efficiency of water and nutrient uptake as well as aeration unlike that of a nonaerated gelled culture. To our knowledge, no previous studies on Al-Taif rose shoot multiplication in bioreactor systems were conducted. However, bioreactor cultures were employed for other rose species. Bayanati and Mortazavi [63] utilized integrated and separate mini-bioreactor systems for shoot multiplication in Rosa hybrid cv. Black Baccara, highlighting that a bioreactor could be used for mass propagation with reduced cost. The growth of Rosa canina cuttings in a small-scale bioreactor with air-lift mode was tested by Kareem et al. [64] for the purpose of producing active compounds. Shoot proliferation and root induction in Rosa damasena and Rosa bourboniana were investigated in a liquid culture system using nodal segments [39]. For the efficient and large-scale induction of roots in microshoots, a rooting vessel was designed and developed to facilitate the micropropagation protocol. Their results highlighted the crucial role of osmotic potential in relation to enhanced growth and development in liquid cultures, vis à vis agar-gelled cultivars, especially in relation to rhizogenesis during micropropagation.
Several studies highlighted the efficiency of liquid/bioreactor culture systems for promoting in vitro shoot growth and proliferation through providing optimal air supply and mixing with oxygen without subjecting the propagules to shear stress [11]. Environmental conditions, including temperature, light intensity, aeration, the amount of air supply, nutrient medium supplementation and mixing, can be more precisely controlled in bioreactor cultures. Additionally, this automated system reduces the cultivation space, decrease the labor cost and facilitates the transfer and harvest of propagules with ease; thus, it can significantly increase the micropropagation effectiveness. Axillary shoot proliferation in Rosaceae, e.g., cherry plum (Prunus cerasifera) [65] and strawberry (Fragaria× ananassa) [66] as well as other plant species, including peace lily (Spathiphyllum cannifolium) [54], cancer bush (Lessertia frutescens) [50] and lacy tree philodendron (Philodendron bipinnatifidum) [67], exhibited better growth in liquid/bioreactor cultures than in gelled cultures.

4. Conclusions

The findings of the present study have demonstrated that the application of GA3 promotes the emergence of buds in Al-Taif roses. BAP exhibited superior efficiency for shoot proliferation compared to other cytokinins, with an optimal concentration of 0.5 mg·L−1. Dark incubation favored shoot proliferation as compared to light incubation. Moreover, symptoms of leaf yellowing and abscission were avoided under dark conditions. Compared with gelled culture, a continuous immersion bioreactor with a net improved axillary shoot number, length and fresh weight. These findings will help improve micropropagation and the in vitro production of Al-Taif rose.

Author Contributions

Conceptualization, A.M.A.-A. and Y.H.D.; methodology, A.M.A.-A. and Y.H.D.; formal analysis, A.M.A.-A.; investigation and data curation, A.M.A.-A. and Y.H.D.; validation, Y.H.D. and R.S.A.-O.; visualization, Y.H.D. and R.S.A.-O.; writing—original draft preparation, A.M.A.-A. and Y.H.D.; writing—review and editing, A.M.A.-A., Y.H.D. and R.S.A.-O. All authors have read and agreed to the published version of the manuscript.

Funding

The authors extend their appreciation to the Deputyship for Research and Innovation “Ministry of Education” in Saudi Arabia for funding this research (IFKSUOR3-094-2).

Data Availability Statement

All data are presented within the article.

Acknowledgments

The authors extend their appreciation to the Deputyship for Research and Innovation “Ministry of Education” in Saudi Arabia for funding this research (IFKSUOR3-094-2).

Conflicts of Interest

The authors declare no conflict of interest.

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Horticulturae 09 01109 g001
Figure 1. In vitro culture establishment of Al-Taif rose: (a) plant material; (b,c) nodal explants used; (d,e) tissue browning; (f) bud break on MS medium containing 3 mg·.L−1 BAP.
Figure 1. In vitro culture establishment of Al-Taif rose: (a) plant material; (b,c) nodal explants used; (d,e) tissue browning; (f) bud break on MS medium containing 3 mg·.L−1 BAP.
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Figure 2. Effect of BAP concentrations and light/dark incubation on number of shoots (a), shoot length (b) and fresh weight (c) of Al-Taif rose explants after 6 weeks. Different letters within a set of values denote significant differences at p ≤ 0.05 according to Tukey’s test.
Figure 2. Effect of BAP concentrations and light/dark incubation on number of shoots (a), shoot length (b) and fresh weight (c) of Al-Taif rose explants after 6 weeks. Different letters within a set of values denote significant differences at p ≤ 0.05 according to Tukey’s test.
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Figure 3. Axillary shoot proliferation of Al-Taif rose under a 16 h light photoperiod and dark conditions ((a,b), respectively); combination of auxins with 0.5 mg·L−1 BAP resulting in leaf yellowing and callus formation under light and dark conditions ((c,d), respectively).
Figure 3. Axillary shoot proliferation of Al-Taif rose under a 16 h light photoperiod and dark conditions ((a,b), respectively); combination of auxins with 0.5 mg·L−1 BAP resulting in leaf yellowing and callus formation under light and dark conditions ((c,d), respectively).
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Figure 4. Photograph showing mass shoot proliferation of Al-Taif rose with limited foliage growth under dark conditions.
Figure 4. Photograph showing mass shoot proliferation of Al-Taif rose with limited foliage growth under dark conditions.
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Figure 5. Length of the main shoot (a), number of shoots (b), and fresh weight (c) of Al-Taif rose in response to culture type (gelled culture and continuous immersion bioreactors with or without net). Different letters show significant differences at p ≤ 0.05.
Figure 5. Length of the main shoot (a), number of shoots (b), and fresh weight (c) of Al-Taif rose in response to culture type (gelled culture and continuous immersion bioreactors with or without net). Different letters show significant differences at p ≤ 0.05.
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Figure 6. (a–c). Effects of culture system (continuous immersion bioreactors with or without net and gelled culture) on Al-Taif rose axillary shoot proliferation in MS medium containing 0.5 mg·L−1 BAP and incubated for 6 weeks under dark conditions.
Figure 6. (a–c). Effects of culture system (continuous immersion bioreactors with or without net and gelled culture) on Al-Taif rose axillary shoot proliferation in MS medium containing 0.5 mg·L−1 BAP and incubated for 6 weeks under dark conditions.
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Table 1. Bud break and growth of Al-Taif rose using nodal explants after 4 weeks in culture.
Table 1. Bud break and growth of Al-Taif rose using nodal explants after 4 weeks in culture.
TreatmentsBud Break
(%)		Number of Days for Bud BreakShoot Length (cm)Fresh Weight
(mg)
Control60 b7.2 a3.9 b54 b
3 mg·L−1 BAP 100 a5.4 b4.8 ab107 a
3 mg·L−1 GA3100 a5.3 b5.8 a72 b
Different letters within a set of values denote significant differences at p ≤ 0.05 according to Tukey’s test.
Table 2. Effects of cytokinin type and concentration on axillary shoot proliferation of Al-Taif rose following 6 weeks in culture.
Table 2. Effects of cytokinin type and concentration on axillary shoot proliferation of Al-Taif rose following 6 weeks in culture.
Cytokinin (mg·L−1)No. Shoots
per Explant		Shoot Length
(cm)		Fresh Weight
per Explant (mg)
MS without PGRs1.4 c4.0 a81 d
BAP   0.51.9 a3.2 bc239 a
1.01.9 a2.8 cd188 ab
2.01.8 a3.0 cd169 abc
2ip   1.01.2 c3.9 a81 d
3.01.5 bc4.2 a130 bcd
5.01.0 c4.0 a94 cd
Kin   1.01.0 c2.8 cd57 d
3.01.3 c2.8 cd69 d
5.01.4 c2.9 cd57 d
TDZ   0.011.0 c3.7 ab90 cd
0.11.2 c 3.6 ab97 cd
0.51.3 c2.6 d79 d
p values
Cytokinin type (A)0.0001 *0.0001 *0.0001 *
Cytokinin Concentrations (B)0.0925 NS0.0860 NS0.5170 NS
A × B0.5510 NS0.0111 *0.6790 NS
Different letters within a set of values denote significant differences at p ≤ 0.05 according to Tukey’s test. NS and * = not significant or significant at p ≤ 0.05, respectively.
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Al-Ali, A.M.; Dewir, Y.H.; Al-Obeed, R.S. Influence of Cytokinins, Dark Incubation and Air-Lift Bioreactor Culture on Axillary Shoot Proliferation of Al-Taif Rose (Rosa damascena trigintipetala (Diek) R. Keller). Horticulturae 2023, 9, 1109. https://doi.org/10.3390/horticulturae9101109

AMA Style

Al-Ali AM, Dewir YH, Al-Obeed RS. Influence of Cytokinins, Dark Incubation and Air-Lift Bioreactor Culture on Axillary Shoot Proliferation of Al-Taif Rose (Rosa damascena trigintipetala (Diek) R. Keller). Horticulturae. 2023; 9(10):1109. https://doi.org/10.3390/horticulturae9101109

Chicago/Turabian Style

Al-Ali, Ali Mohsen, Yaser Hassan Dewir, and Rashid Sultan Al-Obeed. 2023. "Influence of Cytokinins, Dark Incubation and Air-Lift Bioreactor Culture on Axillary Shoot Proliferation of Al-Taif Rose (Rosa damascena trigintipetala (Diek) R. Keller)" Horticulturae 9, no. 10: 1109. https://doi.org/10.3390/horticulturae9101109

APA Style

Al-Ali, A. M., Dewir, Y. H., & Al-Obeed, R. S. (2023). Influence of Cytokinins, Dark Incubation and Air-Lift Bioreactor Culture on Axillary Shoot Proliferation of Al-Taif Rose (Rosa damascena trigintipetala (Diek) R. Keller). Horticulturae, 9(10), 1109. https://doi.org/10.3390/horticulturae9101109

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