Available online at 3rd International Conference on Tissue Engineering, ICTE2013

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1 Available online at Procedia Engineering 59 ( 2013 ) rd International Conference on Tissue Engineering, ICTE2013 Study on effects of different Plant Growth Regulators types in shoot regeneration and node formation of Sutsuki Azalea (Rhododendron indicum): a commercially important bonsai Saiedeh Rahimi a, *, Roohangiz Naderi b, S.A Ghaemaghami, Sepideh Kalatejari, Babak Farham a Department of Agricultural Science, Islamic azad university, Science and Research Branch,Tehran , Iran b Department of Horticultural Science Faculty of Agriculture, University of Tehran, Karaj , Iran Abstract Sutsuki azalea with botanical name Rhododendron indicum is one of the oldest evergreen azaleas, not only considered as a famous ornamental shrub, but also as a medicine to treat disease. Sutsuki azaleas are popular bonsai plants for many reasons. The overall goals of this research are to establish the most suitable cytokinin for in vitro shoot regeneration of Sutsuki azalea. The influence of Plant Growth Regulators was studied on shoot numbers, shoot length and node numbers after 10 weeks. In current experiment, after sterilization the apical shoots contain 4 developing leaf primordia separated and were cultured on ½ Anderson medium with different hormonal treatments. In this stage included three levels of isopentil adenin (2ip) (0, 2, 10) mg/l, Zeatin (0,0/1,0/5) mg/l and Thidiazuron (TDZ) (0, 0/04, 0/2) mg/l that were studied two concentration of hormones with each other. The results showed that 10 mg/l 2ip together with 0/2 mg/l TDZ was the best treatment for increasing the shoot number and 10 mg/l 2ip together with 0/1 mg Zeatin were the best treatment for increasing shoot length and node number. On the basis of available literature, this is the first and significant study regarding the comparative effect of different PGRs on in vitro propagation study of R. indicum. This significant study could be useful for large scale propagation and regeneration of this valuable bonsai The Authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande. * Corresponding author. Tel.: address: biotech.rahimi@yahoo.com The Authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande doi: /j.proeng

2 Saiedeh Rahimi et al. / Procedia Engineering 59 ( 2013 ) Keywords: Sutsuki azalea; Plant growth regulators; Anderson medium. 1. Introduction The sutsuki azaleas (Rhododendron indicum) family Ericaceae as bonsai, grown in the ground or as pot plants. Satsuki evergreen azaleas have been hybridized in Japan for at least 500 years. R.indicum one of the oldest and most famous ever green azaleas, bred since ancient times and they are the type most commonly used for ornamental pruning. In vitro propagation from meristematic tissues is a suitable method for production of Rhododendrons and Azaleas; Their propagation by conventional methods (seeds,cuttings,etc.) is either difficult or infective [20]. The commercial production of ornamental plants is growing worldwide. It is monetary value has significantly increased over the last two decades and there is a great potential for continued further growth in both domestic and international markets [11]. According to a report, in 2004, German in vitro laboratories produced more than 31 million plants of orchids. Other important plant genera are Rhododendron (1.0 million) [21]. One of the major factors affecting the success of in vitro plant propagation is the choice of plant growth regulators (PGRs). As a result, PGRs (especially cytokinins) are often added to culture media for the purpose of controlling different physiological responses in vitro, leading to the production of tissues (such as callus) organs (such as shoots and roots) or whole plants [2]. The development of auxiliary meristems is dependent on the presence of growth regulators in culture medium [10]. Therefore according to reports related to propagation of azalea, typically are used limited levels of different concentrations 2ip, Zeatin and TDZ. Moreover, there are many reports showing that the application of TDZ results in a better shoot regeneration capacity in comparison with other cytokinins [19]. The presence of 1.5 μm TDZ with 2.3 μm Zeatin improved regeneration in evergreen azaleas [8]. Lower cytokinin concentrations have the best effect on shoot regeneration in azaleas. In other research, has reported 5 mg/l Zeatin singly, was the best concentration for the shoot induction evergreen azalea [6]. The objective of study was to find the most effective concentration of growth regulators on shoot proliferation and node formation of Rododendron indicum. 2. Material and Method The study was carried out on Sutsuki azalea (Rhododendron indicum). Azalea plants after flowering stage were collected from the native habitat in Iran. Plants were purchased from Green house-grown plants. Expanded leaves were removed and actively growing shoot tips that their flowers had shed were collected. Shoot tips 15mm in length and containing 4 nodes were used as explants Surface sterilization of explants Explants were surface-sterilized for 5 min in 0.1 % Mercuric chloride and rinsed three times (10 min per rinse) with sterile-deionizer water Culture medium At first, Anderson medium for shoot induction and multiplication was used. In the experiment, ½ Anderson basal medium with 0.01 mg/l indol acetic acid (IAA) were used in total of the treatments. All of the medium in the current work were supplemented with 30 gr sucrose and 7 gr Difco-Bacto agar (sigma A-1296) and the ph was adjusted to 5.8 with 1 N NaOH or 0.1 N HCl prior to autoclaving at 121 c for 30 min Cultivation of explants A total 6 to 9 explants per treatment was used and each three explants were cultivated in a glass jar for 10 weeks. In this research, three different concentrations (0, 2, 10) mg/l isopentil adenin (2ip), (0, 0.1, 0.5) Zeatin and

3 242 Saiedeh Rahimi et al. / Procedia Engineering 59 ( 2013 ) (0, 0.04, 0.2) Thidiazuron (TDZ) totally eighteen treatments were used in the regeneration medium. Each treatment was containing two various concentrations of each cytokinins. Stem explants were cultured on 10 ml of medium contained in glass jars (100 mm 25 mm, 40 ml Volume). All the cultures were incubated at 25±1 c under 16h photoperiod of 2500 LUX light intensity. This experiment was repeated once. Developing shoots longer than 0.5 cm were recorded and the explants were assessed based on shoot number, node number and shoot length after 10 weeks. This research was done based on a randomized complete blocks method in various times of the experiment and data analysis and variance calculations were done using SAS software, then diagrams were plotted. The means were evaluated at the p= 0.05 and 0.01 level of significance using Duncan s multiple range tests. 3. Result and Disscusion We studied the effect of different concentrations of 2ip, Zeatin and TDZ on regeneration of sutsuki azalea (Rhododendron indicum) an ornamental plant. The results are summarized in figure 1, 2 and 3. Our data revealed that there are differences in the effect of the various concentrations of 2ip, Zeatin and TDZ and interaction between them. Shoot tips were excised and transferred on ½ Anderson medium containing IAA (0.01 mg/l) and experiment treatments. Subsequently within the next 10 weeks, differences were observed Effects of cytokinin type and interaction of concentrations on shoot number of sutsuki azalea: Current study showed the positive effect of 2ip with highest concentration on shoot induction and shoot number. The largest number of shoot (4.63) obtained in media containing 10 mg/l 2ip together with 0.2 mg/l TDZ and the lowest shoot number (0.32) was determined in medium supplemented with 0.1 mg/l Zeatin and 0.04 mg/l TDZ (figure.1). In this research, 2ip has been observed to be the most efficient cytokinin for shoot induction of sutsuki azalea. Although, the frequency of axillary bud-break was higher on media containing either Zeatin or TDZ rather than 2ip [8]. The optimal concentrations of TDZ, 2ip depended on Rhododendron genotype and the explants type. For shoot initiation and shoot elongation we used 2 mg/l and 10 mg/l 2ip, which is the treatment concentration level that Briggs [4] has recommended for Azalea explants. Nevertheless, it would be useful to extend concentration ranges of 2ip and TDZ to determine the optimal treatment concentrations for shoot regeneration. Similar our observation, have been reported by others and appear between different cytokinins such as BA, Zeatin, 2ip. The best result was belonging to 2ip with concentration of 10 mg/l and 14 mg/l from leaf explants of Azalea [10]. Previous studies reported that optimal level suggested for using of 2ip has reported 2-10 mg/l that was related to Azaleas species [4]. Similar to other woody plants, 2ip induced a higher proliferation of Rhododendron shoots compared to Zeatin and Kinetin. These results differ from [13],[14]. Interestingly, although 2ip has been reported to be the most commonly used cytokinin for shoot regeneration of Azalea, in this study, the frequency of axillary bud-break was higher on media containing either Zeatin or TDZ rather than 2ip. There were significant interaction between TDZ and 2ip for the number of adventitious shoots. The highest shoot numbers of all sizes formed when 10 mg/l 2ip plus 0.04 mg/l or 0.2 mg/l TDZ were in the medium. Contrary to our finding, the high concentrations of TDZ (especially 20 μm TDZ combined with 25 μm 2ip) were inhibitory to shoot formation [18]. Present study showed the frequency of shoot regeneration and the number of shoots per explants increased with increasing concentrations of TDZ, however, in the present investigation, it was possible to obtain high frequency of shoot organogenesis directly from in vitro derived shoot explants of sutsuki azalea in 2ip-containing medium. The obtained results confirm that adding TDZ together with 2ip to the medium improved adventitious organogenesis similarly as shown for flower explants of Rhododendron L. [19].

4 Saiedeh Rahimi et al. / Procedia Engineering 59 ( 2013 ) Effect of cytokinin type and interaction of concentrations on shoot length and node number: The medium supplemented with 10 mg/l 2ip together with 0.1 mg/l Zeatin resulted in the best shoot length (7.23 mm) and largest number of node (6.34). The lowest shoot length (2.84 mm) and node number (2.56) were found when we used 0.5 mg/l Zeatin together 0.2 mg/l TDZ. Shoot length was significantly affected by the type of cytokinins and their interaction. Presence Zeatin in medium promotes shoot multiplication, although this effect with increasing Zeatin tends to decrease with higher Zeatin concentration (figure 2,3). Increasing two concentrations promotes shoot multiplication independently of explants type, although this effect tends to decrease with higher Zeatin concentration [1]. In accordance with our results, Zeatin and 2ip have been successfully used during shoot proliferation of several Rhododendron species and hybrids [10],[5],[19]. The highest shoot elongation and node formation observed when 10 mg/l 2ip plus 0.1 mg/l Zeatin were in the medium (figure 4). Generally, the higher concentration of 2ip, stimulate shoot regeneration although there was no TDZ or Zeatin in the medium. Nevertheless, the presence of either TDZ or Zeatin in different concentrations was useful for shoot elongation and node formation. On the basis of results obtained in this experiment, 2ip was found to be the most effective cytokinin for shoot elongation and node formation. Following our experiment, explants of Rhododendron fragrantta were transferred to media supplemented with 10 μm 2ip for elongation [7]. Addition of cytokinins to the medium was essential to induce shoot bud regeneration from the explants. Of the three cytokinins tested, 2ip was the most effective. However, when we used only TDZ without 2ip, had showed shoot elongation. This finding are in agreement with result Nayak et al.[16]. In this study, presence of 2ip (10 mg/l and 2 mg/l) improved regeneration in Rhododendron indicum. For Rhododendron.cv. fuchsia, Hsia and Korban [8] reported low shoot induction in the presence of 2ip, while the highest regeneration frequency was induced with TDZ. Some authors Mertens et al. [15]; Hsia and Korban [8]; Vejsadova and Pretova [20] reported TDZ to be an essential growth regulator for the shoot induction of leaf explants. Our results indicate specificity of Rhododendron cultivars to cytokinin 2ip. In accordance with our researches, TDZ was the predominant cytokinin that stimulated the production of adventitious floral structures [18]. Without TDZ and only 2ip, adventitious shoot structures formed, extremely. However when TDZ was present in the medium without 2ip a substantial number of shoot structures was observed and this number tend to decrease when 2ip was the lowest concentration without TDZ. The medium supplemented TDZ has a capacity to inhibit (at least partly) the action of shoots elongation. However, we observed that combination 10 mg/l 2ip together with TDZ caused increase in shoot length and node number. TDZ commonly stimulate shoot proliferation and inhibit their elongation, therefore, inhibition of shoot elongation by TDZ may be consistent with its high cytokinin activity [9],[17]. This research, also showed higher TDZ concentration with together Zeatin significantly stunted the height of newly formed shoots. These findings are in agreement with the results obtained by Tomsone et al.[19] Elongation of shoots was reduced in the medium containing TDZ and Zeatin, moreover shoot derived from explats grown on media containing 0.04 mg/l or higher was compact, and explants developed more callus tissue. Briggs [3] reported a similar observation whereby shoots of deciduous azalea were compact and hyperhydrous on a medium containing 4.5μM TDZ. Moreover, adding TDZ and CPPU to the medium caused formation of hyperhydric explants at high percentages in vitro pear cultivar shoots [12]. Although the addition of Zeatin to TDZ containing media improved shoot quality, increasing the Zeatin concentration from 0.5 to 0.1 mg/l resulted in a significant decrease in the number of proliferating shoots. Similar observation have been reported by Hsia and Korban [8]. However they resulted Zeatin enhanced the shoot length of proliferating shoots, but did not increase shoot multiplication.

5 244 Saiedeh Rahimi et al. / Procedia Engineering 59 ( 2013 ) Fig.1. Influence of cytokinin type and interaction of concentrations on shoot number in Rhododendron indicum Fig.2. Influence of cytokinin type and interactions of concentrations on shoot length in Rhododendron indicum Fig. 3. Influence of cytokinin type and interaction of concentrations on node number in Rhododendron indicum

6 Saiedeh Rahimi et al. / Procedia Engineering 59 ( 2013 ) Fig. 4. The effect of 10 mg/l 2ip plus 0/1 mg/l Zeatin on shoot regeneration and node formation after 10 weeks in culture on ½ Anderson medium 4. Conclusion However, the presence of either TDZ or Zeatin in different concentrations was useful but the highest shoot proliferation observed when 10 mg/l 2ip was in the medium. In general, the higher concentration of 2ip stimulate shoot regeneration and node formation, singly. Acknowledgements The authors are indebted to Iranian Research Organization for Science and Technology (IROST) for the support of project and special thanks Ms. Hourieh Rahimi for her excellent technical assistance. References [1] Almeida, R., Goncalves, S.,Romano, A., In vitro micropropagation of endangered Rhododendron Ponticum L. Subsp. Baeticum (Boissier and Reuter) Handel- Mazzetti. Biodiversity and Conservation 14: [2] Amoo, S.O., Staden, J.V., Influence of plant growth regulators on shoot proliferation and secondary metabolite production in micro propagated Huernia hystrix. Plant Cell Tissue Organ Culture. 112: [3] Briggs, B.A., MCculloch, S.M., Edick, L.A., Micropropagation of azalea using Thidiazuron. Acta Horticulturae, 226: (a,b). [4] Briggs, B.A., MCculloch, S.M., Edicer, L., Micropropagation of azalea (Rhododendron Spp.). Biotechnology in agriculture and forestry, 20: [5] Briggs,B.A., MCculloch, S.M., Caton, L.A., In vitro propagation of Rhododendron. Acta Horticulturae, 364: [6] Chen, X., Xiaodi, H., Goa, H.Y., Haiyan, W., Study on tissue culture of Rhododendron Simsii planch. Journal of Jiangsu forestry Science and Technology. 30: [7] Hebert, C.J., Touchell, D., Ramey, T.G., L ebude, A.V., In vitro Regeneration of Rhododendron fragrantta. Fragrantissimum Improved. SNA Research Conference Vol [8] Hsia, C.N., Korban, S.S., The influence of cytokinins and ionic strength of Anderson s medium on shoot establishment and proliferation of evergreen azalea. Ephytica, 93: [9] Huetteman, C.A., Preece, J.E., Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tissue Organ 33: [10] Iapichino, G., Mcculloch, S., Chen, T.H.H., Adventitious shoot production from leaf explants of Rhododendron. Plant Cell, Tissue and Organ Culture, 30: [11] Jain, S.M., Feeding the world with induced mutations and biotechnology. Proc. Int. Nuclear Conference Global trends and perspectives. Seminar1: Agriculture and bio science. Bangi, Malaysia: MINT; 2002.p [12] Kadota, M., Niimi, Y., Effects of cytokinin types and their concentrations on shoot proliferation and hyperhydricity in in vitro pear cultivar shoots. Plant cell, Tissue and Organ Culture 72: [13] Kamenieka, A., Valka,.J.,Vizarova,G., A comparative study of different cytokinins on the formation of Rhododendron forrestii Balf. F.ex. Diels. Axillary shoots in vitro. Acta physiologiae Plantarum. Vol 20. No.2: [14] Marino, G., Bertazza, G., Micropropagation of Actinidia deliciosa cos Hayward and Tomuri. Scientia Horticulture. 45:

7 246 Saiedeh Rahimi et al. / Procedia Engineering 59 ( 2013 ) [15] Mertens, M., Werbrouck, S., Samyn, G.,Silva, H., Debergh, P.,1996. In vitro regeneration of evergreen azalea from leaves. Plant Cell, Tissue and Organ Culture, 45: [16] Nayak, N.R., Patniak, S., Rath, S.P., Direct shoot regeneration form foliar explants of an epiphytic orchid, Acampe praemorsa (Roxb.) Batter and Mccann. Plant Cell Reports 16: [17] Raghu, A.V.,Geetha, S.P., Balachandran, G.M.I., Ravindran., P.N.,2006. Direct shoot organogenesis from leaf explants of Embelia Ribes Burm. F.; a vulnerable medicinal plant. The Japanese Forest Society and Springer-Verlag Tokyo. 11: [18] Shevade, A., Preece, E., In vitro shoot and floral organogenesis from stamen explants from a Rhododendron PJM groupe clone. Scientia Horticulturae, 56: [19] Tomsone, S., Gertnere, D., Novikova, D., The influence of thidiazuron on shoot regeneration and proliferation of Rhododendron in vitro. Acta Universitatis Latviensis Biology, Vol. 676, pp [20] Vejsadova, H., Growth Regulator effect on in vitro regeneration of Rhododendron cultivars. Horth. Sci. (PRAGUE), 35: [21] Winkelmann, T., Geier, T., Preil, Walter Commercial in vitro plant production in Germany in Plant Cell Tissue Organ Culture. 86: