The Study of Biotechnology and Callus Formation on Rosa damasceana Mill. in the Kashan Region

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1 Proceedings of The Fourth International Iran & Russia Conference 91 The Study of Biotechnology and Callus Formation on Rosa damasceana Mill. in the Kashan Region M.Farhangi-sabet 1 and B.SH.Behboodi 2 1-Atomic Energy Organization of Iran, Nuclear Research Center for Agriculture and Medicine, Nuclear Agriculture Sec.Plant Tissue Culture & Cytogenetic, P.O.Box: 31485/498, Karadj-IRAN, Phone: Fax: &6, mfarhangis@Yahoo.com; 2- Department of Biology, Faculty of science, University of Tehran, phone: , behboodi@khayam.ut.ac.ir ABSTRACT Rosa damascena is cultured in high quantity in Iran (Kashan) and it is one of the most important crops in this area. This species is cultivated for medical, pharmaceutical and alimental plant. This study is carried out for the first time in Iran and is focused on biotechnology and callus formation in this species. we selected six different explants which consisted of:,,,, Petal and Ovary. These explants were cultured on two different basal media: MS and WP with different plant hormones by different concentration: (.1-5mg/l), (.1-5mg/l), IAA(.1-3mg/l), NAA(.1-5mg/l) and 2,4-D(.1-5mg/l). We have introduced best hormones for callus formation result in two media. Monoterpenes were extracted from the culture medium with hexane for 4h and were analyzed by Gas Chromatography-Mass Spectrum. The best result on the callus formation in the MS medium was obtained of stem explant with NAA 1mg/l and 1mg/l.In the WP medium, there was callus formation by petal and ovary explants with NAA.1mg/l and 5mg/l. However, it was resulted that callus formation was in high amount in this species, but most callus lines derived from explants of stem and petal of R.damascena did not produce essential oil at the natural conditions. Keywords: Biotechnology, Callus formation, Plant breeding, Rosa, Tissue culture INTRODUCTION Rosa damascena Mill. is Cultivated in order to produce essence and oil in Bolgharia, India, Turkey and Iran (Bayrak, 1994). High-value aromatic oil is obtained from its petals, that it is used in pharmaceutical and perfume industries (kumar etal, 21). The most expensive rose oils are extracted from petals of R.damascena Mill. and contain 2-phenylethanol as the main component together with geraniol, nerol, citronellol and traces of other volatiles. The main center of cultivation in Iran is Kashan. It is usually is multiplied by cutting and grafting (Rechinger, 1982). Breeding programs in R.damascena Mill. focused on the improvement of various characteristics to enhance the production of flower and pharmaceutical value and essence, including the time length of flowering and the amount of flower per bush and the quality and quantity of essence (Rout etal, 1999). Woody plants have been cultivated under in vitro culture less than herbaceous plants. Until now, about 15 species of it has been successfully micropropagated (Broertjes and Van Horten., 1988). There are a few reports on in vitro propagation of R.damascena Mill. (Khushkhui and sink, 1982; Ishioka and Tanimoto, 199; kumar, 1996; kumar, 21 and Farhangi sabet and SH.behboodi, 23) with limited success. In previous studies on the synthesis and accumulation of monoterpenes by R.damascena cells in vitro, limited success has been achieved (Banthorpe and Barrow, 1983; Banthorpe et al, 1986 and 1988). In this research we studied callus formation of different explants in to both MS and WP media, in various concentrations of auxin and cytokinin hormones. As the result of the following studies the

2 Proceedings of The Fourth International Iran & Russia Conference 92 best culture medium and the best kind of hormone in the most suitable concentration was selected. Also, our present report extends a preliminary study which showed that callus of this species, whilst not accumulating terpenoides. MATERIALS AND METHODS Plant material Seven years old bushes were selected in Barij Essance Company in Kashan area, they were the best species in farming pretection and time length of flowering and amount of buds and crop per hectar. Ten of the best bushes were chosen among all of the bushes and sampled. Sampling was done during late April and May (flowering is generally over by late April - early Jun). Culture establishment In this study, we used six different explants, which consist of apical meristem, lateral meristem, young stem, youny leaf, petal and ovary cm of all explants were neatly cut. Two methods were selected to disinfect explants. The young stem and lateral meristem explants that were scleroticer, were treated with calcium hypochlorite (1%, w/v; 15 min) and the explants were Surface disinfected with an aqueous Solution of mercuric chloride (.4%, w/v; 7min) containing.2% (v/v) Tween 8. These were then repeatedly washed with sterilized distilled water. The young leaf, petal, ovary and apical meristem that were softer, were treated with ethanol (7 ; 1min) and the explants were disinfected with Calcium hypochlorite (5%, w/v; 1 min) containing two drops of Tween 2. There were then repeatedly washed with sterilized distilled water. We used two different basal media MS (Morashige and skoog, 1962) and WP (Lloyd and MC cown., 1981). Different concentrations of auxin and cytokinin hormones were used: (.1-5 mg1-1 ), (.1 5 mg1-1 ), IAA (.1-3 mgl -1 ), NAA (.1 5 mgl -1 ), 2, 4-D (.1-5 mgl -1). The PH of the medium was adjusted to before autoclaving. All Cultures were maintained at 25 ± 1 c under a photoperiod of 14h light and 1h dark cycle. Oil extracts and analyzes Monoterpenes were extracted from calluses with hexane for 4h. Monoterpenes were analyzed by gas liquid chromatography using a glass column (Length: 25m, Phase: CPB5, I.D:.25mm, Type: bonded phase, Material: fused silica, Detector: MS). Injection port temperature and detector temperature were 22 C and 25 C, respectively. Monoterpene products were identified by retention and mass spectral data. Statistical analysis Experiments were repeated at least six times and three explants were cultured in each petridish. The considered factor for studing callus formation was volume of callus. Volume of calluses was obtained by Arashmidous law. We then used Spss 1..5 Software for Calculating means comparisons, analysis of means and analysis of Variance (ANOVA). RESULTS NAA and had the most effect on callus formation among the different groups of hormones that were tested in the MS medium (Fig 1). Also different explants have been studied in all hormone treatments and it was discriminated that the young stem explant is the best callus formatting explant. These results were obtined in NAA 1 mg1-1 and 1 mg1-1 concentrations (Fig 2). Different groups of hormones have been studied by considering the effects on Callus formation in WP medium too. NAA and had the most influence on callus formation (Fig 3). Also different explants have been studied in all hormone treatments that the petal and ovary explants were the best callus formatting explants and these results is

3 Proceedings of The Fourth International Iran & Russia Conference 93 obtained by NAA.1 mgl 1 and 5mg1-1 (Fig 4). Callus lines from petal and stem explant s in MS medium were assayed for total 2- phenylethanol (free and β-d glocoside). The monoterpene components of undifferentiated callus are shown in Fig 6. According to these figures callus lines derived from stem and petal explants did not accumulate 2- phenylethanol under regime studied (Fig 6). DISCUSSION Previous studies on R. damascena, in vitro medium, have focused on micropropagation. According to, khosh-khui and sink (1982) observed that rate of shoot multiplication of R. damascena varied significantly in different Subculture periods. Less shoot proliferation, as well as reduced shoot growth, were observed in Cultures of R.damascena maintained on Supplemented medium (skirvin etal, 199). Similar results have been obtained for shoot regeneration from stem derived callus of R.damascena (Ishioka and Tanimoto, 199). Kumar (1996) showed that R.damascena cultures have less effect on the media with different concentrations of cytokinins and ouxins about shoot multiplication and the proliferation of shoots hardly have been rooted. Also in other studies the comperation of effect between TDZ and on the proliferation and growth of shoots, on the basis of this study TDZ stimulates the growth of shoots (kumar etal, 21). In this research, we have studied callus formation in the MS and WP media. In all pervious researches only MS medium had been used. But we used both MS and WP media. We have approached the Suitable results of callus formation that thay hadn t reported in pervious studies. Suitable callus formation obtained in too media MS and WP. Friable pale yellow cali were typically obtained, but about 1% showed greening with chlorophyll content. Of course the regeneration calluses had difference in color and genus in two MS and WP media, the obtained calluses of MS medium were greener and condenseder (Fig 5). We have researched about the quantity and quality of essence of callus. The above work clearly demonstrates that callus of R.damascena that did not accumulate terpenoids, it is possible that the lack of accumulation in callus is consequence of a lack of storage structures or of general subcellular organization that results in oxidation of these nascent prodacts in a manner precluded in the parent plants. Similar results have been obtained at previous studies (Banthorpe and Barrow, 1983; Banthorpe et al, 1986 and 1988). REFERENCES 1-Banthorpe, D and Barrow, S., Monoterpene Biosynthesis in Extracts from Cultures of Rosa damascene. Phytochemistry, Vol. 22, No, 12, pp Banthorpe, D., Grey, T., Poots, I and Fordham, W., Monoterpene Metabolism in Cultures of Rosa Species. Phytochemistry, Vol. 25, No, 1, pp Banthorpe, D., Branch, S., Poots, I and Fordham, W., Accumulation of 2-Phenylethanol by Callus Derived from Leaf-Bud of Rosa damascene. Phytochemistry, Vol. 27, No, 3, pp Bayrak, A and Akgul, A., Volatile oil composition of Turkish Rose (Rosa damascena). J Sci Food.Agric. 64, Broertjes, C and Van Harten, A.M., Applied mutation breeding for vegetatively propagated crops. In:Developments in crop science,vol.12.elsevier,amsterdam,pp Ishioka, N and Tanimoto,S.,199.Plant regeneration from Bulgarian rose callus. Plant cell Tiss.org.cult.22, Khush-kui, M and Sink, K.C., 1982.Micropropagation of new and old world rose species.j.hort.sci.57, Kumar, A., Studies on in vitro propagation, biochemistry and field evolutions of two economically important plants: Rosa damascena Mil and Gladiolus spp. Thesis, Kumanun University, Nainital, India.

4 Proceedings of The Fourth International Iran & Russia Conference 94 9-Kumar, A., Sood, A., Pali, UT., Gupta, AK and Plani, LS., 21.Micropropagation of Rosa damascena Mill. From nature bushes using thidiazuron. J.Hotr.Sci & Biotech.76(1) Lloyd, G.B and MC Cown, B.H.,1981.Commercially feasible micropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture.proc.natl.plant prop.soc.3, Murashige, T and Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol.15, Rout, G.R., Debata, BK and Das, P., 199.In vitro clonal multiplication of roses.proc.nalt.acad.sci(india).6, Rout, G.R., Samantaray, S., Mottley, J and Das, P., 1999.Biotechnology of the rose: a review of recent progress.sci.hortic.81, Rrchinger, K.H Flora Iranica. Vol 4 15-Skirvin, R.M.199.Rose.In:Ammirato, p.v., Evans, D.R., Sharp,W.R., Bajaj, Y.P.S, Handbook of plant cell culture, Vol 5.McGraw Hill, New york, springer, New york, pp Figures and figure legends Fig 1: Effects of different groups of auxins and cytokinin hormones on callus formation in the MS medium. Volume of calluses ( mm 3 ) ,4 D- 2,4 D- Gropes of Hormones Fig 2: Study of callus formation on the different explants among the different concentration of NAA and in the MS medium.

5 Proceedings of The Fourth International Iran & Russia Conference Volume of calluses (mm 3 ) NAA, 5 Different Concentration of NAA and (mg/l) 3 NAA,3 1 NAA,1 5 NAA,.1 Petal&Ovary Explants Petal&Ovary Fig 3: Effects of different groups of auxins and cytokinin hormones on callus formation in the WP medium Volume of Calluses ( mm 3 ) ,4-D- 2,4-D- Groupes of Hormones Fig 4: Study of callus formation on the different explants among the different concentration of the NAA and in the WP medium.

6 Proceedings of The Fourth International Iran & Russia Conference Volume of Calluses (mm 3 ) NAA, 5 Different Concentration of NAA and (mg/l) 3 NAA,3 1 NAA,1 5 NAA,.1 Petal & Ovary Explants Petal & Ovary Fig 5: A) Callus formation from young stem explant by NAA 1mg/l and 1mg/l in the MS medium. B) Callus formation from young leaf explant by IAA 1mg/l and 3mg/l in the MS medium. C) Callus formation from petal explant by NAA 3mg/l and 3mg/l in the WP medium, there is pink pigmentation on the callus. D) Callus formation from ovary

7 Proceedings of The Fourth International Iran & Russia Conference 97 explant by IAA 2mg/l and 1mg/l in the WP medium (B = 2cm). Fig 6: Essential oil profiles of (A) Stem explant, (B) Petal explant in the undifferentiated calluses.