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AgNO3 improved micropropagation and stimulate in vitro flowering of rose (Rosa x hybrida) cv. Sena

AgNO3 melhora a micropropagação e estimula o florescimento in vitro de rosa (Rosa x hybrida) cv. Sena

Abstract

Rose is one of the most important cut flower in the world. Rose micropropagation was used for production of clonal and disease-free plantlets and to breeding purposes. However, many important rose cultivars showed physiological disorders as early-leaf senescence and very low multiplication rate under in vitro conditions. Our hypothesis is that these symptoms were associated with high sensibility of these cultivars to ethylene accumulation on in vitro environment. The rose cv. Sena was in vitro cultivated under different concentrations of AgNO3 and two light sources, LED and fluorescent lamps, as a way to investigate in vitro similar symptoms to ethylene accumulation. AgNO3 at 1.0-2.0 mg L-1 solved the main in vitro physiological disorders observed in this rose cultivar. Also, AgNO3 stimulated induction of 50% of rose shoots to in vitro flowering at 2.0 mg L-1. Higher concentrations also resulted in flowering induction, but with imperfect flower development.

Keywords:
cut rose; micropropagation; physiological disorders; ethylene inhibitor, flower induction

Resumo

Rosa é uma das flores de corte mais importantes do mundo. A micropropagação de rosas tem sido utilizada para a produção de plântulas clonais e livres de doenças e para fins de melhoramento genético. No entanto, muitas cultivares importantes de rosas apresentaram distúrbios fisiológicos em condições in vitro como senescência foliar precoce e taxa de multiplicação muito baixa. Nossa hipótese é que esses sintomas estão associados à alta sensibilidade dessas cultivares ao acúmulo de etileno no ambiente in vitro. Para o experimento foi escolhida a cultivar de rosa chamada Sena sendo essa cultivada in vitro sob diferentes concentrações de AgNO3, um inibidor da ação do etileno, e duas fontes de luz, LED e lâmpadas fluorescentes, como forma de investigar os sintomas in vitro comumente observados nesta cultivar. O AgNO3 a 1,0-2,0 mg L-1 solucionou os principais distúrbios fisiológicos observados in vitro nesta cultivar rosa. Além disso, o AgNO3 resultou na indução da floração in vitro em 50% das brotações cultivadas in vitro, utilizando a concentração de 2,0 mg L-1. Concentrações mais altas também resultaram em indução de floração, mas com desenvolvimento imperfeito de flores, como exemplo a produção de botões florais sem pétalas.

Palavras-chave:
rosa de corte; micropropagação; desordens fisiológicas; inibidor de etileno; indução de flores

Introduction

Roses (Rosa X hybrida L.) are the most economically and socially important cut flower industry around the world (Rezvanypour and Osfoori, 2011REZVANYPOUR, S.; OSFOORI, M. Effect of chemical treatments and sucrose on vase life of three cut rose cultivars. Journal of Research Agricultural Sciences, v.7, n.2, p.133-139, 2011.), and the main producing countries are Ecuador, Colombia, Kenya, Ethiopia and India, with focus on exportations (Vellekoop, 2018VELLEKOOP, E. A challenging year for the cut rose industry. 2018 Available at : Available at : https://www.floraldaily.com/article/9055298/2018-a-challenging-year-for-the-cut-rose-industry/ , Accessed: July 15th 2020.
https://www.floraldaily.com/article/9055...
). Commercially, rose cultivars are propagated by cutting or grafting. However, there are reports in the literature about the use of micropropagation of different rose cultivars (Kanchanapoom et al., 2010KANCHANAPOOM, K.; SAKPETH, P.; KANCHANAPOOM, K. In vitro flowering of shoots regenerated from cultured nodal explants of Rosa hybrida cv. `Heirloom′. Scienceasia, v.36, n.2, p.161-164. 2010. DOI: https://doi.org/10.2306/scienceasia1513-1874.2010.36.161
https://doi.org/10.2306/scienceasia1513-...
; Jana and Sekhawat, 2011JANA, S.; SEKHAWAT, G.S. Plant growth regulators, adenine sulfate and carbohydrates regulate organogenesis and in vitro flowering of Anethum graveolens. Acta Physiologiae Plantarum, v.33, n.305-311, 2011. DOI: https://doi.org/10.1007/s11738-010-0548-0
https://doi.org/10.1007/s11738-010-0548-...
). Besides micropropagation aiming production of high quality and free-pest and disease plantlets, plant tissue culture has many other applications. As example, in vitro controlled environmental conditions are excellent for studies with in vitro flowering, especially due to the testing isolated factors as plant growth regulators or specific environmental factor, supporting in the study of the biological mechanism of flowering induction and led to development of new technologies, such as in vitro pollination and breeding (Silva et al., 2014SILVA, J.A.T.; ZENG, S.; CARDOSO, J.C.; DOBRÁNZSKI, J.; KERBAUY, G.B. In vitro flowering of Dendrobium. Plant Cell, Tissue and Organ Culture , v.119, p.447-456, 2014. DOI: https://doi.org/10.1007/s11240-014-0561-x
https://doi.org/10.1007/s11240-014-0561-...
).

Flower induction is considered a complex regulation mechanism controlled by both endogenous and environmental factors (Hirata et al., 2016HIRATA, H.; OHNISHI, T.; TOMIDA, K.; ISHIDA, H.; KANDA, M.; SAKAI, M.; YOUSHIMURA, J.; SUZUKI, H.; ISHIKAWA, T.; DOHRA, H.; WATANABE, N. Seasonal induction of alternative principal pathway for rose flower scent. Scientific Reports, v.6, Article 20234, 2016. DOI: https://doi.org/10.1038/srep20234
https://doi.org/10.1038/srep20234...
; Kurokura et al., 2013KUROKURA, T.; MIMIDA, N.; BATTEY, N.H.; HYTÖNEN, T. The regulation of seasonal flowering in the Rosaceae. Journal of Experimental Botany, v.64, n.14, p.4131-4141, 2013. DOI: https://doi.org/10.1093/jxb/ert233.
https://doi.org/10.1093/jxb/ert233...
), hampering studies with isolated factors under greenhouse or field conditions.

In vitro flowering induction has been reported for several species of plants (Panigrahi et al., 2018PANIGRAHI, J.; DHOLU, P.; TANVI, J.; SHAH, T.J.; GANTAIT, S. Silver nitrate-induced in vitro shoot multiplication and precocious flowering in Catharanthus roseus (L.) G. Don, a rich source of terpenoid indole alkaloids. Plant Cell, Tissue and Organ Culture, v.132, n.3, p.579-584, 2018. DOI: https://doi.org/10.1007/s11240-017-1351-z
https://doi.org/10.1007/s11240-017-1351-...
), including different cultivars of rose Rosa x hybrida (Kanchanapoom et al., 2009KANCHANAPOOM, K.; POSAYAPISIT, N.; KANCHANAPOOM, K. In vitro flowering from culture nodal explants of rose (Rosa hybrida L.). Notulae Botanicae Horti Agrobotanici, v.37(2), p.261-263. 2009. DOI: https://doi.org/10.15835/nbha3723077
https://doi.org/10.15835/nbha3723077...
; Kanchanapoom et al., 2010KANCHANAPOOM, K.; SAKPETH, P.; KANCHANAPOOM, K. In vitro flowering of shoots regenerated from cultured nodal explants of Rosa hybrida cv. `Heirloom′. Scienceasia, v.36, n.2, p.161-164. 2010. DOI: https://doi.org/10.2306/scienceasia1513-1874.2010.36.161
https://doi.org/10.2306/scienceasia1513-...
; Vu et al., 2006VU, N.H.; ANH, P.H.; NHUT, D.T. The role of sucrose and different cytokinins in thein vitrofloral morphogenesis of rose (hybrid tea) cv. “First Prize”. Plant Cell Tissue and Organ Culture, v.87, p.315-320, 2006. DOI: https://doi.org/10.1007/s11240-006-9089-z
https://doi.org/10.1007/s11240-006-9089-...
; Zeng et al., 2013ZENG, S.; LIANG, S.; ZHANG, Y.Y.; WU, K.L.; SILVA, J.A.T.; DUAN, J.In vitroflowering red miniature rose. Biologia Plantarum, v.57, p.401-409, 2013. DOI: https://doi.org/10.1007/s10535-013-0306-4
https://doi.org/10.1007/s10535-013-0306-...
). Furthermore, in vitro flowering has a major impact on selective hybridization, especially in the case of pollen use in rare stocks, and may be the first step in the recombination of genetic material through in vitro fertilization (Murthy et al., 2012MURTHY, K.S.R.; KONDAMUDI, R.; CHALAPATI, R.P.V.; PULLAIAH, T. In vitro flowering - a review. Journal of Agricultural Technology, v.8, n.5, p.1517-1536, 2012.) and can be used in flowers breeding programs (Silva et al., 2014SILVA, J.A.T.; ZENG, S.; CARDOSO, J.C.; DOBRÁNZSKI, J.; KERBAUY, G.B. In vitro flowering of Dendrobium. Plant Cell, Tissue and Organ Culture , v.119, p.447-456, 2014. DOI: https://doi.org/10.1007/s11240-014-0561-x
https://doi.org/10.1007/s11240-014-0561-...
).

Khosh-Khui and Silva (2006KHOSH-KHUI, M.; SILVA, J.A.T. In vitro culture of the Rosa species. In: SILVA, J.A.T.D. Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues. Japan: Global Science Books, v.2, Cap. 66, p.514-526, 2006.) reported the potential of in vitro flowering technique for it successful application also for Rosa x hybrida cultivars, in Vietnam. Kanchanapoom et al. (2009KANCHANAPOOM, K.; POSAYAPISIT, N.; KANCHANAPOOM, K. In vitro flowering from culture nodal explants of rose (Rosa hybrida L.). Notulae Botanicae Horti Agrobotanici, v.37(2), p.261-263. 2009. DOI: https://doi.org/10.15835/nbha3723077
https://doi.org/10.15835/nbha3723077...
) observed the in vitro flowering of rose cv. ‘Red Masterpiece’ in 5% of sprouts grown in MS medium containing 2.0 mg L-1 of BA.

Gaseous ethylene normally is associated in most species as inhibitory plant growth regulator for plants flower induction (Iqbal et al., 2017IQBAL, N.; KHAN, N. A.; FERRANTE, A.; TRIVELLINI, A.; FRANCINI, A.; KHAN, M.I.R. Ethylene role in plant growth, development and senescence: interaction with other phytohormones. Frontiers in Plant Science, v.8, p.475, 2017. DOI:https://doi.org/10.3389/fpls.2017.00475
https://doi.org/10.3389/fpls.2017.00475...
). In addition, ethylene, naturally biosynthesized and released by plants, accumulated in in vitro conditions may result in undesirable physiological effects in some species or more sensible-genotypes delaying or restraining the production of high-quality micropropagated plantlets (Cardoso, 2019CARDOSO, J.C. Silver nitrate enhances in vitro development and quality of shoots of Anthurium andraeanum. Scientia Horticulturae, v.253, p.358-363, 2019. DOI: https://doi.org/10.1016/j.scienta.2019.04.054
https://doi.org/10.1016/j.scienta.2019.0...
). In roses cultivated in vitro, symptoms of ethylene accumulation, such as gradual yellowing of leaves were observed by Pratheesh and Kumar (2012PRATEESH, P.T.; KUMAR, A. In vitro flowering in Rosa indica L. International Journal of Pharmacological and Biological Sciences, v.2, n.1, p.196-200, 2012.).

Thus, chemical inhibitors of the ethylene action could be used in culture medium diminishing these symptoms (Pratheesh and Kumar, 2012PRATEESH, P.T.; KUMAR, A. In vitro flowering in Rosa indica L. International Journal of Pharmacological and Biological Sciences, v.2, n.1, p.196-200, 2012.). For instance, products that release or make it available Ag+ ions to plants, such as AgNO3, was reported as main action inhibitor of ethylene, preventing ethylene receptors from binding to it (Yang, 1987YANG, S.F. Regulation of biosynthesis and action of ethylene. Acta Horticulturae, v.201, p.53-60. 1987. DOI: https://doi.org/10.17660/ActaHortic.1987.201.6
https://doi.org/10.17660/ActaHortic.1987...
), improving the quality of micropropagated plantlets (Cardoso, 2019CARDOSO, J.C. Silver nitrate enhances in vitro development and quality of shoots of Anthurium andraeanum. Scientia Horticulturae, v.253, p.358-363, 2019. DOI: https://doi.org/10.1016/j.scienta.2019.04.054
https://doi.org/10.1016/j.scienta.2019.0...
). Silver ion sources are considered anti-ethylene growth regulator and is reported promoting and accelerating flowering in cassava (Manihot esculenta), also showing other phenotypic responses (Hyde et al., 2020HYDE, P.T.; GUAN, X.; ABREU, V.; SETTER, T.L. The anti-ethylene growth regulator silver thiosulfate (STS) increases flower production and longevity in cassava (Manihot esculentaCrantz). Plant Growth Regulation, v.90, p.441-453, 2020. https://doi.org/10.1007/s10725-019-00542-x
https://doi.org/10.1007/s10725-019-00542...
). Also, AgNO3 induced in vitro flowering of different species (Panigrahi et al., 2018PANIGRAHI, J.; DHOLU, P.; TANVI, J.; SHAH, T.J.; GANTAIT, S. Silver nitrate-induced in vitro shoot multiplication and precocious flowering in Catharanthus roseus (L.) G. Don, a rich source of terpenoid indole alkaloids. Plant Cell, Tissue and Organ Culture, v.132, n.3, p.579-584, 2018. DOI: https://doi.org/10.1007/s11240-017-1351-z
https://doi.org/10.1007/s11240-017-1351-...
).

Also, the addition of AgNO3 to the culture medium in specific concentrations may result in significant improvements in the regeneration of in vitro plantlets such as gloxinia (Sinningia speciosa) (Park et al., 2012PARK, E-H.; BAE, H.; PARK, W.T.; KIM, Y.B.; CHAE, S.C.; PARK, S.U. Improved shoot organogenesis of gloxinia (‘Sinningia speciosa’) using silver nitrate and putrescine treatment [online]. Plant Omics, v.5, n.1, p.6-9, 2012. ) and Anthurium andraeanum (Cardoso, 2019CARDOSO, J.C. Silver nitrate enhances in vitro development and quality of shoots of Anthurium andraeanum. Scientia Horticulturae, v.253, p.358-363, 2019. DOI: https://doi.org/10.1016/j.scienta.2019.04.054
https://doi.org/10.1016/j.scienta.2019.0...
).

The other actual and important factor affected in vitro plant regeneration and development are the replacement of conventional tubular fluorescent light by light emitting diodes (LED) (Ramírez-Mosqueda et al., 2017RAMÍREZ-MOSQUEDA, M.A.; IGLESIAS-ANDREU, L.G.; AGUILAR, R.B. The effect of light quality on growth and development of in vitro plantlet of Stevia rebaudiana Bertoni. Sugar Tech, v.19, n.3, p.331-336, 2017. DOI: https://doi.org/10.1007/s12355-016-0459-5
https://doi.org/10.1007/s12355-016-0459-...
; Xu et al., 2020XU, Y.; YANG, M.; CHENG, F.; LIU, S.; LIANG, Y. Effects of LED photoperiods and light qualities on in vitro growth and chlorophyll fluorescence ofCunninghamia lanceolata. BMC Plant Biology, v.20, Article 269, 2020. DOI: https://doi.org/10.1186/s12870-020-02480-7
https://doi.org/10.1186/s12870-020-02480...
). Lighting affects plant development, in part by the changes of endogenous levels of plant hormones, such as cytokinins and ethylene (Zdarska et al., 2015ZDARSKA, M.; DOBISOVÁ, T.; GELOVÁ, Z.; PERNISOVÁ, M.; DABRAVOLSKI, S.; HEJÁTKO, J. Illuminating light, cytokinin, and ethylene signalling crosstalk in plant development. Journal of Experimental Botany , v.66, n.16, p.4913-4931, 2015. DOI:https://doi.org/10.1093/jxb/erv261
https://doi.org/10.1093/jxb/erv261...
)

Based on the several benefits reported by the use of Ag+ sources for in vitro cultivation, the main objective of this study was to evaluate the effects of different concentrations of AgNO3 and the use of LED lighting on the in vitro development of rose cv. Sena. This cultivar presented limited micropropagation due to the presence of physiological disorders similar to those described symptoms due to in vitro accumulation of ethylene.

Materials and Methods

Adult plants, propagated by cutting of rose cultivar ‘Sena’ and under cultivation in greenhouse conditions (35,000 lux and temperature of 15-28 °C) were used as mother plants. Young shoot tips with 1.0 cm length were excised, subjected to surface sterilization and used as explants. The asepsis consisted of immersion of 1-cm shoot tips in alcohol 70% for 30 sec, followed by sodium hypochlorite at 30% (2.0%-2.5% of active chlorine) for 20 min. Subsequently, they were submitted to three washings in autoclaved distilled water, before the shoot tips with approximately 0.3-0.5 mm being excised and inoculated in the culture medium.

The culture medium used for establishment and also for multiplication stage, was the Murashige & Skoog (1962MURASHIGE, T.; SKOOG, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, v.15, p.473-497, 1962. DOI: https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
https://doi.org/10.1111/j.1399-3054.1962...
) with reduction of macronutrients by half (MS½), with addition of 3% sucrose, myo-inositol 0.1 g L-1, and 0.5 mg L-1 Benziladenine, with pH adjusted for 5.8 before addition of agar-agar at 6.5 g L-1. The culture medium was autoclaved at 121°C and 1 kgf cm-2 for 20-min. This culture medium was previously tested in our laboratory conditions with success for establishment stage of rose micropropagation.

After four subcultures in the same culture medium, five in total and using nodal segments of 1.0-1.5 cm length with one leaf, important physiological disorders were observed in in vitro shoots, e.g. low multiplication rate and early-leaf senescence, which resulted in some required modifications of basic culture medium and the experiment with AgNO3.

The MS culture medium was modified by the reduction of KNO3 and NH4NO3 salts by ¼ and CaCl2 increased two-fold from the original concentration of MS, maintaining 3% sucrose, myo-inositol 0.1 g L-1, and 0.5 mg L-1 Benziladenine (BA). There were observed that reduction of nitrogen concentration and increased Calcium could present benefits to in vitro plant development of some woody species (Reed et al. 2013REED, B.M.; WADA, S.; DENOMA, J.; NIEDZ, R.P. Improving in vitro mineral nutrition for diverse pear germplasm. In Vitro Cellular & Developmental Biology - Plant, v.49, p.343-355, 2013. DOI: https://doi.org/10.1007/s11627-013-9504-1
https://doi.org/10.1007/s11627-013-9504-...
; Wada et al., 2015WADA, S.; NIEDZ, R.P.; REED, B.M. Determining nitrate and ammonium requirements for optimal in vitro response of diverse pear species. In Vitro Cellular & Developmental Biology - Plant , v.51, n.1, p.19-27, 2015. DOI: https://doi.org/10.1007/s11627-015-9662-4
https://doi.org/10.1007/s11627-015-9662-...
). To this new modified culture medium for rose in vitro cultivation, different concentrations of AgNO3 added to culture medium were tested: 0, 1, 2, 3, 4 and 5 mg L-1 before pH adjustment. The pH was adjusted for 5.8 for all treatments and before addition of agar-agar at 6.5 g L-1.

Four repetitions (vials) containing five nodal segments each, 0.8-1.0 cm in length, were used for the experiment. Shoots cultivated in culture medium with each AgNO3 concentration were subjected to two sources of light, white fluorescent cold bulbs 40W and to white lighting-emiting diode (LED) bulbs 10W, as a way to test the use of LED light and to evaluate its viability and effects on rose micropropagation. Shoots were cultivated for 60-days in culture medium containing different concentrations of AgNO3. The shoot number and reduction of symptoms related to ethylene accumulation on in vitro conditions were analyzed. The percentage of shoots induced to flowering and with some abnormalities in flower development was realized between the 20 and 60-days of cultivation.

The experiment was conducted under completely randomized design, in a factorial of 6 concentrations of AgNO3 and two types of light sources. The experiment was repeated twice. The data of percentage of flower induction were previously transformed by arcsen√(x+1) and subjected to ANOVA and the means were compared by Tukey’s test at 5% probability using Agroestat software for statistical analysis (Barbosa and Maldonado Junior, 2011BARBOSA, J.C.; MALDONADO JUNIOR, W. AgroEstat- Sistema para análisesestatísticas de ensaios agronômicos. Jaboticabal: FCAV/UNESP, 2011.). The data of percentage of abnormal flowers were subjected to regression analysis and correlated with concentrations of AgNO3.

Results and Discussion

AgNO3 avoids early-leaf senescence in Rosa sp. cv. Sena

The in vitro establishment resulted in successful introduction of rose explants, with low rate of contamination (<10%) and high rate of regenerated shoots (90%) that started the production of new leaves and elongation of stems after 30-d of in vitro inoculation.

The successive subcultures of shoots in the same culture medium increased the number of shoots until the second one. After the third subculture, symptoms of physiological disorders as early leaf senescence and drastic reduction in shoot proliferation propagation resulted in low quality shoot and poor shoot proliferation in 100% of in vitro cultivated shoots (Figure 1A). Symptoms of yellowing leaves were also observed after the first subculture of Rosa indica shoots (Prateesh and Kumar, 2012PRATEESH, P.T.; KUMAR, A. In vitro flowering in Rosa indica L. International Journal of Pharmacological and Biological Sciences, v.2, n.1, p.196-200, 2012.) under similar in vitro conditions used in actual study with cv. Sena.

Figure 1
Effects of addition of AgNO3 in culture medium for in vitro micropropagation of rose cv. Sena: A, early leaf senescence (ln) symptoms observed in rose cultivated in MS plus 0.5 mg L-1 BA; B, normal shoot development under modified MS medium plus BA 0.5 mg L-1 and AgNO3 1.0 mg L-1; C, bud flower induction in shoots of rose cv. Sena in modified MS medium plus BA 0.5 mg L-1 and AgNO3 1.0 mg L-1; D, E, F, main flower abnormalities observed in in vitro flower induction, absence of petals (ap) in bud flowers, bud flower necrosis (bfn) and undeveloped petals (up); G, normal rose flower anthesis on in vitro conditions;

These symptoms observed in rose micropropagation could be associated with the high biosynthesis and/or sensibility of the rose cv. Sena to accumulation of ethylene under in vitro conditions. Ethylene is commonly correlated with flowers senescence, mainly in ornamental plants (Olsen et al., 2015OLSEN, A.; LUTKEN, H.; NYMARK, J.; MULLER, R. Ethylene resistance in flowering ornamental plants - improvements and future perspectives. Horticultural Research, v.2, Article 15038, 2015. DOI: https://doi.org/10.1038/hortres.2015.38
https://doi.org/10.1038/hortres.2015.38...
) and also can cause in vitro physiological disorders, as observed in Habanero peppers (Santana-Buzzy et al., 2006SANTANA-BUZZY, N.; CANTO-FLICK, A.; IGLESIAS-ANDREU, L.G.; MONTALVO-PENICHE, M.C.; LÓPEZ-PUC, G.; BARAHONA-PÉREZ, F. Improvement of in vitro culturing of habanero pepper by inhibition of ethylene effects. HostScience, v.41, n.2, p.405-409. 2006. DOI: https://doi.org/10.21273/HORTSCI.41.2.405
https://doi.org/10.21273/HORTSCI.41.2.40...
) and Anthurium andraeanum (Cardoso, 2019CARDOSO, J.C. Silver nitrate enhances in vitro development and quality of shoots of Anthurium andraeanum. Scientia Horticulturae, v.253, p.358-363, 2019. DOI: https://doi.org/10.1016/j.scienta.2019.04.054
https://doi.org/10.1016/j.scienta.2019.0...
).

In rose micropropagation, the major peak of ethylene biosynthesis occurred after 4-8 d of culture in MS culture medium, but the authors did not observe any plant symptoms caused by ethylene accumulation in the cv. Madame Georges Delbard’, since when the ACC precursor of ethylene was added (Gaspar et al., 1989GASPAR, T.; KEVERS, C.; BOUILLENE, H.; MAZIERE, Y.; BARBE, J-P. Ethylene production in relation to rose micropropagation through axyllary budding. p.302-312, 1989. In: Biochemical and physiological aspects of ethylene production in lower and higher plants: Proceeding… Belgium. DOI: https://doi.org/10.1007/978-94-009-1271-7_34
https://doi.org/10.1007/978-94-009-1271-...
). However, early leaf senescence associated with apical necrosis also was observed in another cultivar of rose, called ‘Starina’, mainly when exposed to Indoleacetic acid (IAA) in rooting stage (Podwyszynska and Hempel 1988PODWYSZYNSKA, M.; HEMPEL, M. The factors influencing acclimatization of Rosa hybrida plants multiplied in vitro to greenhouse conditions. Acta Horticulturae, v.226, p.639-642, 1988. DOI: https://doi.org/10.17660/ActaHortic.1988.226.88
https://doi.org/10.17660/ActaHortic.1988...
; Podwyszynska and Goszczynska 1998PODWYSZYHSKA, M.; GOSZCZYFISKA, D.M. Effect of inhibitors of ethylene biosynthesis and action, as well as calcium and magnesium on rose shoot rooting, shoot-tip necrosis and leaf senescence in vitro. Acta Physiologiae Plantarum , v.20, n.1, p.91-98, 1998. DOI: https://doi.org/10.1007/s11738-998-0049-6
https://doi.org/10.1007/s11738-998-0049-...
). In addition, leaf yellowing was observed after first subculture on in vitro shoots of Rosa indica grown in MS medium added 0.5 mg L-1 IAA and 1 mg L-1 BA (Pratheesh and Kumar, 2012PRATEESH, P.T.; KUMAR, A. In vitro flowering in Rosa indica L. International Journal of Pharmacological and Biological Sciences, v.2, n.1, p.196-200, 2012.). These results showed a genotype-dependent response of ethylene-sensibility in rose, with recurrent and similar symptoms observed with actual study in rose cv. Sena.

Similarly, in actual study, the use of AgNO3 reduced drastically the symptoms observed on in vitro shoot culture of rose cv. Sena (Figure 1B). The shoots obtained in AgNO3-added culture medium resulted in green coloration of stems and leaves (3-5/shoot), good shoot proliferation (2.5-2.8 shoots/explants) and absence of the in vitro previous symptoms reported for this cultivar, compared with culture medium without AgNO3 (0.7-1.0 shoots/explants and no green leaves in shoots). These results showed that Ag+ ions, added to the culture medium, was efficient to reduce the in vitro negative symptoms in shoots of rose cv. Sena, such as ealy leaf yellowing and senescence associated with reduced multiplication rates, showing that the main cause was due to ethylene accumulation in the in vitro conditions and the high ethylene-sensibility of this rose cultivar to this plant hormone, which resulted in poor in vitro development.

AgNO3 influenced induction and development of in vitro flowers in rose cv. ‘Sena’

Since at very low concentration of 1.0 mg L-1, AgNO3 added to the culture medium resulted in flowering induction of rose cv. Sena, (Table 1; Figure 1C-G).

Table 1
Effects of light-source and concentrations of AgNO3 added in the culture medium on leaf senescence-abortion and on in vitro flowering induction of rosa cv. ‘Sena’

Panigrari et al. (2018) also observed that addition of 0.1 µM of AgNO3 also resulted in flowering induction of Catharanthus roseus, but the flowering induction in this species was only observed when AgNO3 salt was combined with high concentrations of cytokinins (3 mg L-1 of BA and 3 mg L-1 Kinetin) in the culture medium. Also, in culture medium used for rose micropropagation BA was used at 0.5 mg L-1 as cytokinin source, which may have contributed for this interaction BA x Ag ions, this last acting as action repressor of ethylene.

The maximum percentage of flowering-induced shoots (50%) was observed at 2.0 mg L-1 concentration (Table 1). The use of major concentrations reduced or maintained the percentage of shoots with flowers, but increased the number of bud flowers and flowers with different types of abnormalities (Figure 1C-E; Figure 2).

Figure 2
Regression analysis of correlation between AgNO3 concentration added to the culture medium and the percentage of abnormal flowers of Rosa x hybrida developed in vitro. *significative at 5% probability.

These results showed two different response of AgNO3 according to the stage of flowering, the induction phase and the flowers development. The stage of flower induction and flower development has different requirements, caused by differential gene expression (Hong and Jackson, 2015HONG, Y.; JACKSON, S. Floral induction and flower formation-the role and potential application of miRNAs. Plant Biotechnology Journal, v.13, p.282-292, 2015. DOI: https://doi.org/10.1111/pbi.12340
https://doi.org/10.1111/pbi.12340...
).

In this study addition of Ag+ ions promoted the flowering induction in rose shoots, but high concentrations resulted in possible toxic effects to flower development. Silver ions acts causing block in ethylene receptors in plants, inhibiting ethylene effects (Santana-Buzzy et al., 2006SANTANA-BUZZY, N.; CANTO-FLICK, A.; IGLESIAS-ANDREU, L.G.; MONTALVO-PENICHE, M.C.; LÓPEZ-PUC, G.; BARAHONA-PÉREZ, F. Improvement of in vitro culturing of habanero pepper by inhibition of ethylene effects. HostScience, v.41, n.2, p.405-409. 2006. DOI: https://doi.org/10.21273/HORTSCI.41.2.405
https://doi.org/10.21273/HORTSCI.41.2.40...
; Faria et al., 2017FARIA, G.A.; FELIZARDO, L.M.; FERREIRA, A.F.A.; ROCHA, P.S.; SUZUKI, A.N.; SOUZA, A.S.; JUNGHANS, T.G.; COSTA, M.A.P.C.; PEIXOTO, A.P.B; MORAIS, A.R.; LOPES, B.G.; OLIVEIRA, T.A. Concentrations of silver nitrate in the in vitro development and conservation of Passiflora gibertii N.E. Brown. American Journal of Plant Sciences, v.8, p.2944-2955, 2017. DOI: https://doi.org/10.4236/ajps.2017.812199
https://doi.org/10.4236/ajps.2017.812199...
). Thus, ethylene may be involved directly or indirectly in inhibiting of the flowering induction in roses. The response of floral transition to ethylene was species-dependent, but in Arabidopsis, ethylene delayed the flowering induction and is considered as inhibitor (Achard et al., 2007ARCHARD, P.; BAGHOUR, M.; CHAPPLE, A.; HEDDEN, P.; VAN DER STRAETEN, D.; GENSCHIK, P.; MORITZ, T.; HARBERD, N.P. The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristem-identity genes. Proceedings of the National Academy of Sciences, v.104(15), p.6484-6489, 2007. DOI: https://doi.org/10.1073/pnas.0610717104
https://doi.org/10.1073/pnas.0610717104...
).

In roses, the response of flowering induction seems to be multi-factorial, but there were some reports with in vitro flower induction of roses (mean 20% of plantlets) with culture medium containing BA (Dobres et al., 1998DOBRES, M.; WILLIAMS, L.; GAIL, R. Micropropagation of rose plants. United States patent 5, v.843, p.782 December 1. 1998.), showing that cytokinins could be an important PGR for rose flower induction. Similarly, maximum in vitro flower bud induction in rose cv. ‘Orange Parade’ was reported by Wang et al. (2002WANG, G.Y.; YUAN, M.F.; HONG, Y. In vitro flower induction in roses. In Vitro Cellular & Developmental Biology - Plant , v.38, n.5, p.513-518, 2002. DOI: https://doi.org/10.1079/IVP2002340
https://doi.org/10.1079/IVP2002340...
) using the cytokinins TDZ at 0.5 mg L-1(49.2%) or Zeatine at 0.5 mg L-1 (44.2%), combined with NAA at 0.1 mg L-1. The use of major cytokinins concentration did not result in increases percentage of bud flower induction. These authors also reported that flowering induction in rose is highly genotype-dependent, with a range of in vitro bud flower induction from 17.3% to 50% depending on the genotype.

Interestingly, results of our actual study using modified MS medium, only the addition of cytokinin BA at 0.5 mg L-1 did not result in any shoots with flowers, while Wang et al. (2002WANG, G.Y.; YUAN, M.F.; HONG, Y. In vitro flower induction in roses. In Vitro Cellular & Developmental Biology - Plant , v.38, n.5, p.513-518, 2002. DOI: https://doi.org/10.1079/IVP2002340
https://doi.org/10.1079/IVP2002340...
) observed 12.5% bud flower of rose cv. Orange Parade induction using this same concentration of BA. Only addition of AgNO3 resulted in rose cv. Sena in vitro flower induction and bud flower development (Table 1; Figure 1).

Cytokinins were reported as inhibitor of ethylene biosynthesis and flower senescence, while ethylene accelerates this physiological response in rose flowers (Wu et al., 2017WU, L.; MA, N.; JIA, Y.; ZHANG, Y.; FENG, M.; JIANG, C-Z.; MA, C.; GAO, J. An ethylene-induced regulatory module delays flower senescence by regulation cytokinin content. Plant Physiology, v.173, n.1, p.853-862, 2017. DOI: https://doi.org/10.1104/pp.16.01064
https://doi.org/10.1104/pp.16.01064...
), suggesting antagonistic effects of cytokinins and ethylene, at least for flower senescence. These results suggests that cytokinins and AgNO3 could act together on flowering induction of rose cultivars, cytokinins acting reduced biosynthesis and Ag+ ions reduced the sensibility of rose shoots to ethylene under in vitro conditions, which led to flowering induction of rose shoots. Other studies with cytokinins x ethylene interactions were very limited (Iqbal et al., 2017IQBAL, N.; KHAN, N. A.; FERRANTE, A.; TRIVELLINI, A.; FRANCINI, A.; KHAN, M.I.R. Ethylene role in plant growth, development and senescence: interaction with other phytohormones. Frontiers in Plant Science, v.8, p.475, 2017. DOI:https://doi.org/10.3389/fpls.2017.00475
https://doi.org/10.3389/fpls.2017.00475...
). Pratheesh and Kumar (2012PRATEESH, P.T.; KUMAR, A. In vitro flowering in Rosa indica L. International Journal of Pharmacological and Biological Sciences, v.2, n.1, p.196-200, 2012.) also reported the in vitro flowering of Rosa indica, but authors observed only one bud flower in a single shoot at 50 mg L-1 of AgNO3, while in this paper 50% of shoots produced bud flowers at 2.0 mg L-1 of AgNO3.

Another interesting phenomenon observed in in vitro flower-induced rose shoots is the formation of abnormal flowers, which increases frequency as AgNO3 concentrations increase (Figure 1, Figure 2). Similar results were observed in Anthurium andraeanum, where concentrations above 2.0 mg L-1 of AgNO3 resulted in non-desirable effects (Cardoso, 2019CARDOSO, J.C. Silver nitrate enhances in vitro development and quality of shoots of Anthurium andraeanum. Scientia Horticulturae, v.253, p.358-363, 2019. DOI: https://doi.org/10.1016/j.scienta.2019.04.054
https://doi.org/10.1016/j.scienta.2019.0...
). These symptoms observed in rose and anthurium could be a result of plant phytotoxicity action by accumulation of high concentrations of Ag ions in plant tissues, similar to reported for tobacco (Cvjetko et al., 2018CVJETKO, P.; STEFANIC, P.P.; ZOVKO, M.; BIBA, R.; TKALEC, M.; DOMIJAN, A-M.; VRCEK, I.V.; LETOFSKY-PAPST, I.; SIKC, S.; BALEN, B. Phytotoxic effects of silver nanoparticles in tobacco plants. Environmental Science Pollution Research, v.25, n.6, p.5590-5602, 2018. DOI: https://doi.org/10.1007/s11356-017-0928-8
https://doi.org/10.1007/s11356-017-0928-...
).

Ethylene also has important effects on flower development. Ethylene was associated with petal cell expansion (Liu et al., 2013LIU, D.; LIU, X.; MENG, Y.; SUN, C.; TANG, H.; JIANG, Y.; KHAN, M.A.; XUE, J.; MA, N.; GAO, J. An organ-specific role for ethylene in rose petal expansion during dehydration and rehydration. Journal of Experimental Botany , v.64, n.8, p.2333-2344, 2013. DOI: https://doi.org/10.1093/jxb/ert092
https://doi.org/10.1093/jxb/ert092...
) and recent studies have associated ethylene as a regulator of homeotic genes associated with flower development (Iqbal et al., 2017IQBAL, N.; KHAN, N. A.; FERRANTE, A.; TRIVELLINI, A.; FRANCINI, A.; KHAN, M.I.R. Ethylene role in plant growth, development and senescence: interaction with other phytohormones. Frontiers in Plant Science, v.8, p.475, 2017. DOI:https://doi.org/10.3389/fpls.2017.00475
https://doi.org/10.3389/fpls.2017.00475...
). These affirmatives were coincident with the results of this study, as the main abnormalities observed was the non-developed petals in the bud flowers (>70% of the total abnormalities observed) (Figure 1D), while another abnormality consisted of bud flower necrosis (18.5%, Figure 1E) or defected petals (Figure 1F).

The prevalent color of in vitro petals was pink (Fig. 1G), while in greenhouse conditions red color of flowers characterize the cultivar. Also, the number of petals obtained in vitro is reduced compared to normal flowers under greenhouse with the same cultivar.

The type of light has low influence on in vitro shoot development and flowering induction in rose cv. ‘Sena’. According to Terfa et al. (2013TERFA, M.T.; SOLHAUG, K.A.; GISLEROD, H.R.; OLSEN, J.E.; TORRE, S. A high proportion of blue light increases the photosynthesis capacity and leaf formation rate of Rosa x hybrida but not affect the time to flower opening. Physiologia Plantarum, v.148, p.146-159. 2013. DOI: https://doi.org/10.1111/j.1399-3054.2012.01698.x
https://doi.org/10.1111/j.1399-3054.2012...
) rose was irradiance-dependent flowering, but the comparison between LED (80% Red; 20% Blue) and high-pressure sodium (low blue) lamps did not result in differences in flowering for Rosa x hybrida cv. Toril. In this study, the use of white LEDs (10W potency) reduced the percentage of abnormal flowers obtained in vitro and could be used for rose micropropagation, with similar results to white fluorescent tubular lamps (40W potency), reducing the costs with electrical energy for micropropagation.

The link between flowering in rose and ethylene was discussed, and two main hypotheses could be formulated with actual experiment, the hypothesis of inhibition of flowering induction by ethylene in rose and the importance of ethylene for flower petal development. In vitro flowering also has physiological interest, helping to understand the mechanism of rose flowering, but could also be used for breeding purposes, using early in vitro flowering for techniques such as in vitro pollination and fertilization (Zulkarnain et al., 2015ZULKARNAIN, Z.; TAPINGKAE, T.; TAJI, A. Applications of in vitro techniques in plant breeding. In: Al-Khayri, J.; Jain, S.; Johnson, D. (eds) Advances in plant breeding strategies: breeding, biotechnology and molecular tools. Switzerland: Springer, Cham., 2015. p.293-328.).

Conclusions

The use of AgNO3 in rose micropropagation increased the shoot proliferation and quality of shoots by avoiding physiological disorders observed in Ag-free culture medium, confirming the sensibility of rose cv. Sena to in vitro ethylene accumulation. In addition, AgNO3 added to the culture medium also induced in vitro flowering of rose cv. ‘Sena’, which could be used for better comprehension of flowering physiology in rose species, and could be applied for emerging technologies, as in vitro breeding.

Acknowledgements

JCC thanks to CNPQ for project number 311083/2018-8. AVCSM (Process number 2018/02595-5), BSO (Process number 2018/07122-8) and MEBSO (Process number 2019/00243-7) thanks FAPESP for their scholarships.

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  • Area Editor: Fernanda Carlota Nery

Author Contribution

  • JCC: contributes with the main idea, installation and conduction of the experiments, data analysis, writing and final edition of the paper, AVCSM, BSO and MEBSO contribute with data collection, software statistical analysis and paper writing.

Publication Dates

  • Publication in this collection
    23 Nov 2020
  • Date of issue
    Jan-Mar 2021

History

  • Received
    17 Mar 2020
  • Accepted
    07 Oct 2020
  • Published
    05 Nov 2020
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