Impaginato 105 Adv. Hort. Sci., 2020 34(1): 105­111 DOI: 10.13128/ahsc­8406 Indirect shoot organogenesis and in vitro root formation of Antirrhinum majus L. by using of sodium nitroprusside M.S. Rezaei Zafarghandi 1, M. Rahmati­Joneidabad 2 (*) 1 Department of Agronomy and Plant Breeding Science, College of Aburaihan, University of Tehran, Tehran‐Pakdasht, Iran. 2 Department of Horticultural Science, Faculty of Agriculture, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran. Key words: Callus, nitric oxide, plant growth regulator, Snapdragon. Abstract: The aim of this study was to determine the effect of different concen­ trations of sodium nitroprusside (SNP) on in vitro shoot organogenesis from hypocotyl explant derived from in vitro grown seedling as well as root forma­ tion of Antirrhinum majus L. (Snapdragon). In the first experiment, different concentrations of 2,4­dichlorophenoxyacetic acid (2,4­D) (0, 2.26, 4.52, and 6.79 μM) were used for callus formation. The highest callus fresh weight (1.86 g) as well as callogenesis frequency (93.34%) were observed in Murashige and Skoog (MS) medium containing 4.52 μM 2,4­D. In the later experiments, various concentrations (0, 10, 20, 30, 40, and 50 μM) of sodium nitroprusside (SNP) were applied for shoot regeneration from callus that derived from hypocotyl segments. Based on our results, MS medium supplemented with 4.44 μM 6­ benzylaminopurine (BAP) plus 0.49 μM 3­indolebutyric acid (IBA) along with 30 μM SNP had the highest shoot organogenesis frequency (93.34%) and shoot number (6.33) from callus. In root induction experiment, different concentra­ tions (0, 20, 40, 60, 80, and 100 μM) of SNP were applied and MS medium con­ taining 60 μM SNP was the best treatment for root induction. The survival rate of plantlets was more than 95% in acclimatization stage. The present study describes an efficient regeneration system for Snapdragon. 1. Introduction Snapdragon (Antirrhinum majus L.) is known as one of the most signifi­ cant ornamental plants which has worldwide values as cut flowers, herba­ ceous landscape plants, and flowering potted plants (El­Nashar, 2017). Also, snapdragon has high commercial values with its wide range of color, shape, structure, and size (Weiss et al., 2016). The commercial propaga­ tion of snapdragon is via seeds. The seed propagation cannot ensure the whole genetic uniformity so seed­propagated plants may indicate unde­ sired phenotypes, quality, and regeneration potential. Therefore, plants (*) Corresponding author: rahmati@asnrukh.ac.ir Citation: REZAEI ZAFARGHANDI M.S., RAHMATI­JONEIDA­ BAD M., 2020 ­ Indirect shoot organogenesis and in vitro root formation of Antirrhinum majus L. by using of sodium nitroprusside. ­ Adv. Hort. Sci., 34(1): 105­111. Copyright: © 2020 Rezaei Zafarghandi M.S., Rahmati­ Joneidabad 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 25 March 2019 Accepted for publication 31 October 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(1): 105­111 106 might be selected randomly without taking necessary care. These features of seeds exert a negative impact on sexual production of this plant (Jaworski et al., 2016). Therefore, the development, as well as improvement of in vitro culture techniques in this ornamental plant, is of high paramount (Hesami and Daneshvar, 2016). A rapid regeneration pathway for A. majus could be useful for commercial propagation of nursery and cut­flower industries as well as breed­ ing programs (Sheyab et al., 2010). Also, in vitro cul­ ture of this plant is necessary for producing high quality/price ratio flower. Moreover, genetic engi­ neering by using biolistic or Agrobacterium methods could be known as a viable alternative in traditional breeding methods for developing distinguished snap­ dragon cultivars in order to satisfy market demands (Davies et al., 2013). On the other hand, the efficien­ cy of gene transformation in snapdragon, obtained via these methods, remains low in this ornamental plant because of the lack of efficiency in vitro propa­ gation protocols (Azadi et al., 2016). According to the previous study, Sheyab et al. (2010) indicated that high applicability of transformation in A. majusis completely depended on the propagation proce­ dures. Therefore, the use of in vitro culture for pro­ ducing Snapdragon could reduce these problems that occur in the commercial production of this plant (Atkinson et al., 1989; Sheyab et al., 2010). Adjusting the culture medium with suitable plant growth regulators (PGRs) in various combinations and concentrations could enhance the propagation potential of various genotypes and explants (Hesami et al., 2017 a, b; Jafari et al., 2017; Hesami et al., 2018 a, b, c; Hesami and Daneshvar, 2018 a, b; Hesami et al., 2019 a, b, c). Thus, it is significant to improve the propagation protocols by using suitable PGRs in order to overcome difficulties associated with clonal regeneration and gene transformation strategies to satisfy the increasing demand for A. majus (Newbury, 1986; Sheyab et al., 2010; Hesami and Daneshvar, 2016). Nitric oxide is known as a messenger molecule for regulating plant development (Neill et al., 2003; Hesami et al., 2019 d). This molecule has recently been characterized as one of the phytohormones (Leterrier et al., 2012). Nitric oxide is known as a ubiquitous bioactive molecule that mainly con­ tributed to various plant developmental processes such as fruit ripening, flowering, organ senescence, and germination (Jimenez­Quesada et al., 2017). The exterior usage of nitric oxide might improve the tol­ erance of plants under various stresses such as tem­ perature, heavy metals, ultraviolet radiation, drought, and salinity (Laspina et al., 2005; Qiao and Fan, 2008). The activation rate of nitric oxide has been evaluated by the exogenous usage of sodium nitroprusside instead of using NO gas directly because of some technical difficulties (Sarropoulou and Maloupa, 2017). In recent years, nitric oxide is used for developing in vitro plant propagation (Rico­ Lemus and Rodríguez­Garay, 2014). Kalra and Babbar (2010) indicated that nitric oxide could enhance the regeneration response via increasing the number of meristems and recommended that nitric oxide regu­ lates the gene expression related to differentiation of meristems. Also, Sarropoulou and Maloupa (2017) recommended that nitric oxide exert a powerful impact on cell division and also it could be involved in shoot organogenesis and proliferation. Han et al. (2009) and Sarropoulou et al. (2014) showed that in vitro shoot proliferation as well as root formation of plantlets were promoted significantly by applying SNP to the MS medium in Malus hupehensis and cherry rootstocks, respectively. Although there are few studies about the effect of nitric oxide on improving in vitro shoot organogenesis (Han et al., 2009; Xu et al., 2009; Kalra and Babbar, 2010; Tan et al., 2013; Sarropoulou et al., 2014; Arun et al., 2017; Ghadakchiasl et al., 2017; Sarropoulou and Maloupa, 2017), there is no research evidence on the effect of this molecule on shoot organogenesis of snapdragon. Thus, the aim of this study was to evaluate the effect of sodium nitroprusside (SNP) on indirect shoot organogenesis as well as root formation that derived from hypocotyl explants of snapdragon in order to reduce the time of in vitro shoot propagation. 2. Materials and Methods The seeds of snapdragon were washed under tap water for 30 min. Further surface sterilization treat­ ments were conducted in a laminar airflow chamber. The seeds were surface sterilized with 70% ethanol for 10 seconds and soaked for 10 min in 10% (v/v) NaOCl. Afterward, the seeds were washed three times in sterilized distilled water. Subsequently, the sterilized seeds were inoculated on one­tenth strength MS medium. After 8­10 days, seeds were germinated, and the hypocotyl segment from in vitro seedling was used as a source of explant for the latter experiment. The MS medium containing 3% (w/v) sucrose, 0.6% (w/v) agar was used as basal medium. The basal Rezaei Zafarghandi and Rahmati‐Joneidabad ‐ Indirect shoot organogenesis of Antirrhinum majus 107 medium was fortified with different PGRs, and pH 5.8 adjusted with 1 N NaOH before autoclaving at 121°C for 20 min. All growth regulators except sodium nitroprusside (SNP) were added before autoclaving. SNP was added after autoclaving by filtering. All cul­ tures were maintained at 25±2°C with 55­60% rela­ tive humidity, and 16 h photoperiod (65 μmol m­2 s­1) that provided by cool white fluorescent light. Hypocotyl explants (0.5­1.0 cm) from 1­week­old in vitro seedlings (Fig. 1 a) were inoculated on MS medium supplemented with various concentrations (0, 2.26, 4.52, and 6.79 μM) of 2,4­D for callus forma­ tion. All of the culture vessels were kept at 25±2°C in the absence of light. Data of callus formation fre­ quency (%) and callus fresh weight (g) were mea­ sured after four weeks of culture. Calli were cultured in the regeneration medium containing 4.44 μM BAP plus 0.49 μM IBA supple­ mented with different SNP concentrations (0, 10, 20, 30, 40, and 50 μM). The shoots regeneration fre­ quency and the number of shoots per callus were determined after 5 weeks of treatment. Shoots with 0.5­1.5 cm in length were transferred to MS medium supplemented with 1 mg/l GA3 (elonga­ tion medium) for 4 weeks. Then, the elongated shoots (2­3 cm elongation) were chosen and transferred to the half strength MS medium containing 3% (w/v) sucrose, 0.6% (w/v) agar and different concentrations (0, 20, 40, 60, 80, and 100 μM) of SNP. Rooting per­ centage (%) and root number including main and sec­ ondary roots were evaluated after 30 days. Plantlets with well­developed root system were removed from the media, washed thoroughly with sterile water and transplanted into potting mixture containing autoclaved perlite and cocopeat mixture (1:1) and covered with transparent plastic to maintain high humidity. The plastic sheets were removed after 4 weeks in order to acclimatize plantlets to green­ house condition, and the plants were shifted to pots comprising garden soil. All experiments were performed with a total of 10 replicates per treatment and were repeated 3 sets. The data were analyzed by ANOVA using SAS version 9.3 followed by Duncan’s multiple range test (DMRT, P<0.05). 3. Results and Discussion The callogenesis experiment was conducted in order to figure out the most suitable and efficient concentration of 2,4­D for callus formation. The result of this study indicated that the maximum per­ cent of callus induction (93.34%) and callus weight (1.86 g) (Fig. 2) were achieved on MS medium con­ taining 4.52 μM 2,4­D (Fig. 1b). In the agreement with our result, Sangwan and Harada (1975) showed that acceptable callus formation of Snapdragon through stem explant was achieved in MS medium containing 4.52 μM 2,4­D. The earlier study proved the positive effect of 2,4­D on the callus formation, Fig. 1 ­ In vitro shoot regeneration through indirect organogene­ sis from seedling derived hypocotyl segments of Antirrhinum majus L. (a) Seedling from in vitro seed ger­ mination; (b) Yellow­greenish and friable callus induction on MS + 4.52 μM 2,4­D; (c) Shoot regeneration from cal­ lus on MS medium containing 4.44 μM BAP plus 0.49 μM IBA along with 30 μM SNP; (d) In vitro root formation on MS + 60 μM SNP; (e) Acclimatized regenerated plants after four weeks. Fig. 2 ­ Effect of different concentrations of 2,4­Din MS medium on (a) callus formation frequency and (b) callus fresh weight of A. majus. Means followed by the same letter are not significantly different at P<0.05 as determined by Duncan’s multiple range test; Vertical bars: standard error. Adv. Hort. Sci., 2020 34(1): 105­111 108 and also this study reported that 2,4­D may be involved in endogenous IAA metabolism regulation by inducing some specific proteins and controlling DNA methylation (Pan et al., 2010). However, in another study, the callus formation of A. majus via hypocotyl explant was obtained on MS medium with different concentrations of NAA plus 10% coconut milk (Atkinson et al., 1989). By increasing sodium nitroprusside from 10 μM to 30 μM, shoot regeneration was improved (Fig. 3). Also, the maximum frequency of shoot organogene­ sis (93.34%) and shoots number (6.33) were observed in MS medium supplemented with 30 μM SNP (Fig. 1c, Fig. 3). However, the higher level (more than 30 μM) of sodium nitroprusside might limit the shoots number and shoot organogenesis frequency. Calli can grow in MS medium supplemented with 50 μM sodium nitroprusside. These obtained results rec­ ommended that sodium nitroprusside can promote shoot organogenesis in proper doses. Our results indicated that sodium nitroprusside completely pro­ moted shoot organogenesis from hypocotyl seg­ ments in MS medium along with 4.44 μM BAP plus 0.49 μM IBA. Thus, BAP and SNP appear to have a synergistic effect on shoot regeneration. The effect of NO on in vitro organogenesis is completely associat­ ed with cytokinins (Arun et al., 2017). It has previous­ ly been shown that NO might interact with auxin and cytokinin, linking the regulation of cell division to dif­ ferentiation during the de­differentiation and re­dif­ ferentiation of plant cells (Ghadakchiasl et al., 2017; Karalija et al., 2017). Tun et al. (2001) observed that NO plays a potential role in mediating plant hormone (auxin and cytokinin) signal transduction during growth and development. Carimi et al. (2005) found that BA stimulates the release and accumulation of NO in plant suspension cell cultures. Therefore, in the present study, SNP may have functioned as an inter­ mediary for adventitious shoot differentiation and regeneration, as suggested by Han et al. (2009) in Malus hupehensis. Our results showed that MS medium supplement­ ed with 1 mg/l GA3 caused shoot elongation. NO (pre­ cursor of SNP) has been reported to influence several plant developmental events in which gibberellins (GAs) play crucial roles such as seed germination, hypocotyl elongation, acquisition of photomor­ phogenic traits, and primary root growth (Beligni and Lamattina, 2000). However, the actual interaction between NO and GAs has been described for only a limited number of these physiological events. In fact, most of our current knowledge of the mechanisms underlying the interplay between GAs and NO is restricted to the regulation of seed germination (Neill et al., 2003) and the inhibition of hypocotyl elonga­ tion during seedling de­etiolation (Lozano­Juste and León, 2011). NO has been described as acting upstream of GAs (Bethke et al., 2007), regulating both biosynthesis and perception/transduction of GAs (Lozano­Juste and León, 2011). There was no root formation in the MS medium without sodium nitroprusside while adding SNP pro­ moted root formation significantly. By increasing the concentration of SNP from 0 to 60 μM, the root for­ mation frequency (100%) and roots number (8.33) (Fig. 4) were increased significantly (Fig. 1d). However, the roots number was decreased when the SNP level was over 60 μM. Root formation is known as the meristematic development of tissues after removing the primary root system (Dash et al., 2017; Jafari et al., 2017). It was indicated that nitric oxide was involved in the response of auxins during root induction in cucumber (Pagnussat et al., 2003) and another report demonstrated that a NO­mediated cGMP dependent pathway was involved in this process (Pagnussat et al., 2003). In order to form the root meristem, auxins promoted parenchyma cells dedifferentiation and entrance to cell division (Klerk et al., 1995; Fujita and Syono, 1996). Also, Gouvea et Fig. 3 ­ Effect of different concentrations of SNP in MS medium containing 4.44 μM BAP plus 0.49 μM IBA on (a) regene­ ration frequency and (b) shoot number of A. majus. Means followed by the same letter are not significantly different at P<0.05 as determined by Duncan’s multiple range test; Vertical bars: standard error. Rezaei Zafarghandi and Rahmati‐Joneidabad ‐ Indirect shoot organogenesis of Antirrhinum majus 109 Plantlets that had well­developed roots were transferred successfully into small pots consisting of perlite and cocopeat mixture (1:1). Our results showed that the rooted plants had 95% survival rate in the acclimatization stage. Afterwards, within 20 days after transferring plantlets to the greenhouse, the normal growth of plantlets was resumed (Fig. 1e). Similar to our results, Hesami and Daneshvar (2016) indicated that by acclimatization of the snap­ dragon plantlets in the perlite and cocopeat mixture (1:1), 90% survival rate was obtained. In conclusion, we have developed a method for indirect shoot organogenesis from hypocotyl explants of A. majus. It is of note that SNP, a donor of NO, has a direct effect on in vitro shoot differentia­ tion and rooting of the snapdragon explants. SNP may interact with auxin and cytokinin, linking the regulation of cell division to cell differentiation during the dedifferentiation and redifferentiation of plant cells. The improvement of ornamental plant by con­ ventional methods (hybridization, inbreeding and mass selection) is time and labor consuming, depends on the existing gene pool(s) and violently influenced by environmental conditions. On the other hand, callus culture can be utilized as a power­ ful tool for genetic cell transformation via somaclonal variation and promoting mutagenesis and genetic engineering that can be either more rapid than tradi­ tional breeding and leading to new genes and geno­ types. The indirect plant regeneration system devel­ oped for A. majus provided a step towards the appli­ cation of such methodology, for this ornamental plant. Moreover, this protocol is rapid with induction of callus to acclimatizing of plantlets to greenhouse completed within 21 weeks. References ARUN M., NAING A.H., JEON S.M., AI T.N., AYE T., KIM C.K., 2017 ­ Sodium nitroprusside stimulates growth and shoot regeneration in chrysanthemum. ­ Hort. Environ. Biotech., 58(1): 78­84. ATKINSON N.J., FORD­LLOYD B.V., NEWBURY H.J., 1989 ­ Regeneration of plants from Antirrhinum majus L. cal‐ lus. ­ Plant Cell Tissue Organ Cult., 17(1): 59­70. AZADI P., BAGHERI H., NALOUSI A.M., NAZARI F., CHAN­ DLER S.F., 2016 ­ Current status and biotechnological advances in genetic engineering of ornamental plants. ­ Biotechnol. Adv., 34(6): 1073­1090. BELIGNI M.V., LAMATTINA L., 2000 ­ Nitric oxide stimulates seed germination and de‐etiolation, and inhibits hypocotyl elongation, three light‐inducible responses in al. (1997) suggested that the role of nitric oxide in signal transduction pathways for root elongation is similar to the role of auxins in this step. Therefore, it became clear that nitric oxide might have an interac­ tion with auxins in regulating cell division to differen­ tiation in ‘‘de­differentiation’’ and ‘‘re­differentia­ tion’’ steps of plant cells (Ötvös et al., 2005). The pos­ itive effect of nitric oxide on improving root induction is reported in various species (Huang and She, 2003; Correa­Aragunde et al., 2004; Han et al., 2009). Sarropoulou et al. (2014) recommended that nitric oxide could (a) produce an antioxidant condition that protects auxins from deteriorations as well as oxida­ tion, (b) speed up cell expansion in order to improve rooting in plants, (c) serve as a downstream messen­ ger in the IAA signaling pathway, (d) regulate enzyme activities or cell­cycle genes that are associated with auxin signal transduction, and (e) reduce the lignifica­ tion of cell wall. By using exogenous sodium nitro­ prusside, the root induction in mung bean was pro­ moted significantly (Huang and She, 2003). Furthermore, sodium nitroprusside can induce root hair induction in lettuce (Lombardo et al., 2006), and development of lateral roots in tomato (Correa­ Aragunde et al., 2004). Fig. 4 ­ Effect of different concentrations of SNP in MS medium on (a) root formation frequency and (b) root number of A. majus. Means followed by the same letter are not significantly different at P<0.05 as determined by Duncan’s multiple range test; Vertical bars: standard error. 110 Adv. Hort. Sci., 2020 34(1): 105­111 plants. ­ Planta, 210(2): 215­221. BETHKE P.C., LIBOUREL I.G., AOYAMA N., CHUNG Y.­Y., STILL D.W., JONES R.L., 2007 ­ The Arabidopsis aleu­ rone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy. ­ Plant Physiology, 143(3): 1173­1188. CARIMI F., ZOTTINI M., COSTA A., CATTELAN I., DE MICHELE R., TERZI M., LO SCHIAVO F., 2005 ­ NO sig‐ nalling in cytokinin‐induced programmed cell death. ­ Plant Cell Environ., 28(9): 1171­1178. CORREA­ARAGUNDE N., GRAZIANO M., LAMATTINA L., 2004 ­ Nitric oxide plays a central role in determining lateral root development in tomato. ­ Plant, 218(6): 900­905. DASH M., YORDANOV Y.S., GEORGIEVA T., TSCHAPLINSKI T.J., YORDANOVA E., BUSOV V., 2017 ­ Poplar PtabZIP1‐like enhances lateral root formation and bio‐ mass growth under drought stress. ­ The Plant J., 89(4): 692­705. DAVIES K.M., DEROLES S.C., BOASE M.R., HUNTER D.A., SCHWINN K.E., 2013 ­ Biolistics‐based gene silencing in plants using a modified particle inflow gun. ­ Biolistic DNA Delivery: Methods and Protocols, pp. 63­74. EL­NASHAR Y., 2017 ­ Response of snapdragon (Antirrhinum majus L.) to blended water irrigation and arbuscular mycorrhizal fungi inoculation: uptake of minerals and leaf water relations. ­ Photosynthetica, 55(2): 201­209. FUJITA H., SYONO K., 1996 ­ Genetic analysis of the effects of polar auxin transport inhibitors on root growth in Arabidopsis thaliana. ­ Plant Cell Physiol., 37(8): 1094­ 1101. GHADAKCHIASL A., MOZAFARI A.­A., GHADERI N., 2017 ­ Mitigation by sodium nitroprusside of the effects of salinity on the morpho‐physiological and biochemical characteristics of Rubus idaeus under in vitro condi‐ tions. ­ Physiol. Mol. Biol. Plants, 23(1): 73­83. GOUVEA C., SOUZA J., MAGALHAES A., MARTINS I., 1997 ­ NO‐releasing substances that induce growth elongation in maize root segments. ­ Plant Growth Regul., 21(3): 183­187. HAN X., YANG H., DUAN K., ZHANG X., ZHAO H., YOU S., JIANG Q., 2009 ­ Sodium nitroprusside promotes multi‐ plication and regeneration of Malus hupehensis in vitro plantlets. ­ Plant Cell Tiss. Organ Cult., 96(1): 29­34. HESAMI M., DANESHVAR M.H., 2016 ­ Regeneration from callus which is produced from cotyledon of Antirrhinum majus. ­ Indo­Am. J. Agric. Vet. Sci., 4(1): 20­24. HESAMI M., DANESHVAR M.H., 2018 a ­ In vitro adventi‐ tious shoot regeneration through direct and indirect organogenesis from seedling‐derived hypocotyl seg‐ ments of Ficus religiosa L.: an important medicinal plant. ­ HortScience, 53(1): 55­61. HESAMI M., DANESHVAR M.H., 2018 b ­ Indirect organo‐ genesis through seedling‐derived leaf segments of Ficus religiosa ‐ a multipurpose woody medicinal plant. ­ J. Crop Sci. & Biotech., 21(2): 129­136. HESAMI M., DANESHVAR M.H., LOTFI A., 2017 a ­ In vitro shoot proliferation through cotyledonary node and shoot tip explants of Ficus religiosa L. ­ Plant Tiss. Cult. & Biotech., 27(1): 85­88. HESAMI M., DANESHVAR M.H., YOOSEFZADEH­NAJAFABA­ DI M., 2018 a ­ Establishment of a protocol for in vitro seed germination and callus formation of Ficus religiosa L., an important medicinal plant. ­ Jundishapur J. Nat. Pharm Prod., 13(4): e62682. HESAMI M., DANESHVAR M.H., YOOSEFZADEH­NAJAFABA­ DI M., 2019 d ­ An efficient in vitro shoot regeneration through direct organogenesis from seedling‐derived petiole and leaf segments and acclimatization of Ficus religiosa. ­ J. Forestry Res., 30(3): 807­815. HESAMI M., NADERI R., TOHIDFAR M., 2019 a ­ Modeling and optimizing in vitro sterilization of Chrysanthemum via multilayer perceptron‐non‐dominated sorting genetic algorithm‐II (MLP‐NSGAII). ­ Frontiers in Plant Sci., 10: 282. HESAMI M., NADERI R., TOHIDFAR M., YOOSEFZADEH­ NAJAFABADI M., 2019 b ­ Application of adaptive neuro‐fuzzy inference system‐non‐dominated sorting genetic algorithm‐II (ANFIS‐NSGAII) for modeling and optimizing somatic embryogenesis of Chrysanthemum. ­ Frontiers in Plant Sci., 10: 869 HESAMI M., NADERI R., YOOSEFZADEH­NAJAFABADI M., 2018 b ­ Optimizing sterilization conditions and growth regulator effects on in vitro shoot regeneration through direct organogenesis in Chenopodium quinoa. ­ BioTechnologia, 99(1): 49­57. HESAMI M., NADERI R., YOOSEFZADEH­NAJAFABADI M., MALEKI M., 2018 c ­ In vitro culture as a powerful method for conserving Iranian ornamental geophytes. ­ BioTechnologia, 99(1): 73­81. HESAMI M., NADERI R., YOOSEFZADEH­NAJAFABADI M., RAHMATI M., 2017 b ­ Data‐driven modeling in plant tissue culture. ­ J. Appl. Environ. Biol. Sci., 7(8): 37­44. HESAMI M., TOHIDFAR M., ALIZADEH M., DANESHVAR M.H., 2019 c ­ Effects of sodium nitroprusside on callus browning of Ficus religiosa: an important medicinal plant. ­ J. Forestry Res., 31: 1­8. HUANG A., SHE X., 2003 ­ Effect of nitroprusside (SNP) on the generation of adventitious roots in mung bean hypocotyl cuttings. ­ Acta Bot. Boreal­Occident Sin., 23: 2196­2199. JAFARI M., DANESHVAR M.H., LOTFI A., 2017 ­ In vitro shoot proliferation of Passiflora caerulea L. via cotyle‐ donary node and shoot tip explants. ­ BioTechnologia, 98(2): 113­119. JAWORSKI C.C., THÉBAUD C., CHAVE J., 2016 ­ Dynamics and persistence in a metacommunity centred on the plant Antirrhinum majus: theoretical predictions and an empirical test. ­ J. Ecol., 104(2): 456­468. JIMENEZ­QUESADA M.J., CARMONA R., LIMA­CABELLO E., TRAVERSO J.Á., CASTRO A.J., CLAROS M.G., DE DIOS Rezaei Zafarghandi and Rahmati‐Joneidabad ‐ Indirect shoot organogenesis of Antirrhinum majus 111 ALCHÉ J., 2017 ­ Generation of nitric oxide by olive (Olea europaea L.) pollen during in vitro germination and assessment of the S‐nitroso‐and nitro‐proteomes by computational predictive methods. ­ Nitric Oxide, 68: 23­37. KALRA C., BABBAR S.B., 2010 ­ Nitric oxide promotes in vitro organogenesis in Linum usitatissimum L. ­ Plant Cell Tiss. Organ Cult., 103(3): 353­359. KARALIJA E., ZELJKOVIĆ S.Ć., TARKOWSKI P., MURATOVIĆ E., PARIĆ A., 2017 ­ The effect of cytokinins on growth, phenolics, antioxidant and antimicrobial potential in liquid agitated shoot cultures of Knautia sarajevensis. ­ Plant Cell Tiss. Organ Cult., 131(2): 347­357. KLERK G.­J.D., KEPPEL M., BRUGGE J.T., MEEKES H., 1995 ­ Timing of the phases in adventitious root formation in apple microcuttings. ­ J. Exp. Bot., 46(8): 965­972. LASPINA N., GROPPA M., TOMARO M., BENAVIDES M., 2005 ­ Nitric oxide protects sunflower leaves against Cd‐induced oxidative stress. ­ Plant Sci., 169(2): 323­ 330. LETERRIER M., VALDERRAMA R., CHAKI M., AIRAKI M., PALMA J.M., BARROSO J.B., CORPAS F.J., 2012 ­ Function of nitric oxide under environmental stress con‐ ditions, pp. 99­113. ­ In: KHAN N.A., R. NAZAR, N. IQBAL, and N.A. ANJUM (eds.) Phytohormones and abi‐ otic stress tolerance in plants. Springer, Berling, Gemany, pp. 308. LOMBARDO M.C., GRAZIANO M., POLACCO J.C., LAMATTI­ NA L., 2006 ­ Nitric oxide functions as a positive regula‐ tor of root hair development. ­ Plant Signal Behav., 1(1): 28­33. LOZANO­JUSTE J., LEÓN J., 2011 ­ Nitric oxide regulates DELLA content and PIF expression to promote photo‐ morphogenesis in Arabidopsis. ­ Plant Physiol., 156(3): 1410­1423. NEILL S.J., DESIKAN R., HANCOCK J.T., 2003 ­ Nitric oxide signalling in plants. ­ New Phytol., 159(1): 11­35. NEWBURY H., 1986 ­ Multiplication of Antirrhinum majus L. by shoot‐tip culture. ­ Plant Cell Tissue Organ Cult., 7(1): 39­42. ÖTVÖS K., PASTERNAK T.P., MISKOLCZI P., DOMOKI M., DORJGOTOV D., BOTTKA S., DUDITS D., FEHÉR A., 2005 ­ Nitric oxide is required for, and promotes auxin‐medi‐ ated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures. ­ Plant J., 43(6): 849­860. PAGNUSSAT G.C., LANTERI M.L., LAMATTINA L., 2003 ­ Nitric oxide and cyclic GMP are messengers in the indole acetic acid‐induced adventitious rooting process. ­ Plant Physiol., 132(3): 1241­1248. PAN Z., ZHU S., GUAN R., DENG X., 2010 ­ Identification of 2, 4‐D‐responsive proteins in embryogenic callus of Valencia sweet orange (Citrus sinensis Osbeck) follow‐ ing osmotic stress. ­ Plant Cell Tiss. Organ Cult., 103(2): 145­153. QIAO W., FAN L.M., 2008 ­ Nitric oxide signaling in plant responses to abiotic stresses. ­ Integr. Plant Biol., 50(10): 1238­1246. RICO­LEMUS M., RODRÍGUEZ­GARAY B., 2014 ­ SNP as an effective donor of nitric oxide for in vitro plant cell and tissue culture. ­ J. Plant Biochem. Physiol., 2(3): 127­ 128. SANGWAN R., HARADA H., 1975 ­ Chemical regulation of callus growth, organogenesis, plant regeneration, and somatic embryogenesis in Antirrhinum majus tissue and cell cultures. ­ J. Exp. Bot., 26(6): 868­881. SARROPOULOU V., DIMASSI­THERIOU K., THERIOS I., 2014 ­ Ιn vitro plant regeneration from leaf explants of the cherry rootstocks CAB‐6P, Gisela 6, and MxM 14 using sodium nitroprusside. ­ In Vitro Cell Dev. Biol. Plant, 50(2): 226­234. SARROPOULOU V., MALOUPA E., 2017 ­ Effect of the NO donor “sodium nitroprusside” (SNP), the ethylene inhibitor “cobalt chloride” (CoCl2) and the antioxidant vitamin E “α‐tocopherol” on in vitro shoot proliferation of Sideritis raeseri Boiss. & Heldr. subsp. raeseri. ­ Plant Cell Tiss. Organ Cult., 128(3): 619­629. SHEYAB S., SHATNAWI M.A., SHIBLI R.A., OBEIDAT M., AL­ SHADAIDEH A.N., ALHUSSAEN K.M., ABU­ZAHRA T., 2010 ­ Micro propagation and medium term conserva‐ tion of Antirrhinum majus L. ­ Jordan J. Agric. Sci., 6(2): 171­182. TAN B.C., CHIN C.F., ALDERSON P., 2013 ­ Effects of sodium nitroprusside on shoot multiplication and regeneration of Vanilla planifolia Andrews. ­ In Vitro Cell Dev. Biol. Plant, 49(5): 626­630. TEWARI R.K., KIM S., HAHN E.­J., PAEK K.­Y., 2008 ­ Involvement of nitric oxide‐induced NADPH oxidase in adventitious root growth and antioxidant defense in Panax ginseng. ­ Plant Biotechnol. Rep., 2(2): 113­122. TUN N.N., HOLK A., SCHERER G.F., 2001 ­ Rapid increase of NO release in plant cell cultures induced by cytokinin. ­ FEBS letters, 509(2): 174­176. WEISS J., MÜHLEMANN J.K., RUIZ­HERNÁNDEZ V., DUDAREVA N., EGEA­CORTINES M., 2016 ­ Phenotypic space and variation of floral scent profiles during late flower development in Antirrhinum. ­ Front Plant Sci., 7: 1­12. XU J., YIN H., WANG W., MI Q., LIU X., 2009 ­ Effects of sodium nitroprusside on callus induction and shoot regeneration in micropropagated Dioscorea opposita. ­ Plant Growth Regul., 59(3): 279­285.