Influence of medium on growth and development of wild rose in vitro Monika Tkalec 1, Nada Parađiković 1, Svjetlana Zeljković 2, Tomislav Vinković 1 1 Faculty of Agriculture Osijek, Trg Svetog Trojstva 3, 31 000 Osijek, Hrvatska (nparadj@pfos.hr) 2 Faculty of Agriculture Banja Luka, Bulevar Vojvode Petra Bojovića 1A, 78 000 Banja Luka, Republika Srpska, BiH Abstract This research was conducted during 2010 in controlled environmental conditions as two step production of wild rose rootstock. The aim of this study was to determine growth and development of wild rose at two different growth media, to preserve genotype characteristics and to shorten a multiplication time of virus-free plant material. Plants were cultivated on two different growth media. The first medium contained two hormones BAP (6-Benzylaminopurine) and IBA (Indole-3-butyric acid), and the second contained just hormone BAP. Plants that were grown on these growth media were transplanted in commercial substrate and treated with biostimulant Radifarm by watering. Investigated parameters were shoot number, stem height, root length and stem and root mass. The results showed that addition of BAP hormone alone in the growth medium resulted in significantly longer stems, but it had no influence on shoot number. Application of the biostimulant had positive effect on shoot number and root weight. Investigation shows how the biostimulant application in R. canina transplants production improves growth and development of root and above-ground mass which is important for faster plant adaptation on stress during transplanting. Key words: Wild roses, in vitro, growth medium, biostimulant, substrate Introduction The usage of Rosa canina plant is various therefore it found its place in culinary, decorating, cosmetic industry, horticulture and as medicinal plant. The most commonly used part of the plant for its medicinal properties is the fruit which are well known to contain a large amount of vitamin C which is antioxidant (Daels-Rakotoarison et al., 2002; Kilicgun et al., 2009). Thus it is considered to be an astringent, carminative, diuretic, laxative, ophthalmic and tonic. Besides being used for its medicinal properties, wild roses found a place in floriculture as rootstock for grafting or breeding of cultivated species of roses. A modern method of tissue culture provides faster reproduction of wild rose s rootstocks and thus shortened breeding time to one year. The aim of this study was to determine growth and development of wild rose at two different growth media, to preserve genotype characteristics and to shorten a multiplication time of virus-free plant material. Material and methods The investigation was conducted during 2009 and 2010 in the laboratory at the Agriculture Faculty in Banja Luka, Bosnia-Herzegovina, as two step production of wild rose rootstock. Investigation started on 5 th of November 2009 by transplanting explants of wild rose on two different growth media. The experiment consisted of 20 flasks of one variant (BI) arranged in four repetition and 20 flasks of other 104
variant. The first variant of growth medium contained hormone BAP (6-benzyl aminopurine) in the concentration of 0.5 mg/l and hormone IBA (Indole-butyric acid) in the concentration of 0.1 mg/l, while the other variant of growth medium contained only the BAP hormone in concentration of 0.5 mg/l. Flasks were kept in a chamber under controlled conditions at temperature of 20 C and under artificial illumination, sixteen hours of light («day») and eight hours of darkness («night»). When plants consumed all medium for their growth and development, they were transplanted in containers with substrate of manufacturer Fruhstorfer Erde, Premium Blumenerde. Before transplanting the plants were immersed in a 0.3% solution of fungicides based on captan to ensure resistance to the disease after transplantation. Forty plants from each variant (BI and B) were transplanted in four repetitions representing treatment and control. with biostimulant Radiafarm in the concentration of 0,25% was carried out immediately after the transplantation and each following week. Radiafarm belongs to a group of biostimulants containing glucosides (energy growth factors) and amino-acids (arginine and asparagine) which stimulate root development. A month later each of the plants were transplanted in a separate container where its development and Radiafarm treatment was continued. During the transplantation the number of plants that have failed to adapt to substratum was recorded. When the plants were sufficiently developed for permanent transplantation, stem and root length and mass were recorded. Research was performed as a split-split-plot experiment with factors: A) growths medium and B) treatment. Following parameters were examined in this study: influence of different growth media and biostimulants on shoot number, stem height and weight and root length and mass. Acquired results were statistically processed using variance analysis with the SAS 9.0 program. Results and discussion Statistical analysis of the data showed that the growth medium did not significantly affect the properties of shoots. However, the number of shoots recorded on plants grown on medium with BAP was 19.17% higher than plants grown on the second medium. According to Pati et. al (2005), Arnold et al. (1995) microshoots of R. kordesii cvs. John Franklin and Champlain rooted well in MS medium with low or no auxin. Also, inclusion of BAP (1.0 10.0 mg/l) in the culture medium was essential for bud break and shoot multiplication of R. hybrida (Hasegawa, 1980; Wulster and Sacalis, 1980). Bressan et al. (1982) reported max- imum promotive effects using BAP as compared with. 2-isopentyladenine (2-ip). The highest number of shoots on average 2.61 per plant was recorded on plants that were grown on the medium with BAP hormone alone, while the lowest number of shoots per plant 1.99 was recorded on plants grown on the medium containing both hormones, BAP and IBA. The treatment with biostimulants significantly affected the number of shoots (p = 0.05). Interaction between the properties of growth medium and treatment with biostimulator showed statistical significance (p = 0.01). The root mass was significantly higher in the medium with just BAP hormone than in the medium containing both the BAP and IBA (p = 0.01). The treatment with biostimulant also showed a significant difference of root mass compared to control plants (p = 0.01). The greatest root mass was observed on plants grown on the medium with only BAP hormone and treated with the biostimulant (21.010 g), while the smallest root mass was observed in plants grown on the medium containing both BAP and IBA hormones (15.670 g), and belonged to the control (Table 1). The root mass of plants grown on the medium with BAP hormone was 17.94% greater than the root mass of the plants grown on the medium with both hormones. Plants treated with the biostimulant had 13.96% greater root mass than control plants. Similar results were presented by Vinković et al. (2009). in whose research the largest root mass of tomato were recorded in plants treated with biostimulant Radifarm. 105
Influence of medium on growth and development of wild rose in vitro In the study of Parađiković et al. (2009) the annual flower types treated with biostimulant Radifarm gave greater weight of seedlings compared to untreated. Table 1. Influence of the hormones and the biostimulant on shoot number and root fresh mass of wild rose Variant BI* (A1) 2,392 1,990 2,191 15,670 17,548 16,609 B** (A2) 2,550 2,675 2,612 18,163 21,010 19,586 2,471 2,332 2,4015 16,916 19,279 18,097 0,01 ns ns 0,9965 0,05 ns 0,1306 0,5553 0,01 0,8996 1,1542 ns 0,05 0,4901 0,7619 ns BI* - Gowth medium with BAP (6-benzil aminopurine) and IBA (Indole-3-butyric acid) hormones B** - growth medium with BAP (6-benzil aminopurine) hormone ns = non significant In this study, stem length was significantly influenced by the growth medium containing just BAP hormone (p = 0.01), while the treatment with biostimulant did not significantly influence this property. Maximum stem length, 18.5 cm, therefore was recorded on plants grown on the medium with BAP hormone, while the minimum stem length, 15.8 cm, was recorded on plants grown on the growth medium containing both BAP and IBA hormones (Table 2). Stem length in plants grown on the medium with BAP hormone was 12.45% higher than the plants grown on the medium with both hormones. Significantly higher stem length was recorded on control plants grown on the medium with just BAP hormone compared to control plants grown on the medium with hormones BAP and IBA (p = 0.05). Stem mass was significantly influenced by the growth media with BAP hormone (p = 0.01), while the treatment with the biostimulant did not significantly influence this property. The highest stem weight 47.983 g was recorded on the plants grown on the medium containing only BAP hormone and belonged to the treatment with the biostimulant, while the lowest stem weight 30.960 g was recorded on plants grown on the medium with BAP and IBA hormone and belonged to the control. Plants grown on the growth medium containing just BAP hormone had 35.98% greater stem mass than the once grown on the medium with both hormones. Plants treated with the biostimulant had 12.82% greater stem mass than the control plants. Significantly higher stem weight was recorded in control plants grown on the growth medium with BAP compared to control plants grown on the growth medium with BAP and IBA hormones (p = 0.01). Root length was not significantly influenced by either the hormone or the biostimulant. 106
Table 2. Influence of the hormones and the biostimulant on stem length and stem fresh mass of wild rose Variant Stem length Stem mass BI* (A1) 15,890 16,142 16,016 30,960 36,850 33,905 B** (A2) 17,523 18,500 18,011 44,230 47,983 46,106 16,706 17,321 17,013 37,595 42,416 40,005 0,01 9,4723 ns ns 0,05 5,1602 ns ns 0,01 1,7989 ns ns 0,05 0,9800 ns ns BI* - Gowth medium with BAP (6-benzil aminopurine) and IBA (Indole-3-butyric acid) hormones B** - growth medium with BAP (6-benzil aminopurine) hormone ns = non significant Conclusion The results of investigation showed better development of plants that were cultivated on the growth medium with BAP hormone alone, as those plants produced greater root and stem weight and greater stem height. Application of the biostimulant had positive effect on shoot number and root weight. Investigation shows how the biostimulant application in R. canina transplants production improves growth and development of root and above-ground mass which is important for faster plant adaptation on stress during transplanting. References Bressan PH, Kim YJ, Hyndman SE, Hasegawa PM, and Bressan RA. (1982) Factors affecting in vitro propagation of rose. J. Amer. Soc. Hortic. Sci., 107, 979-990. Daels-Rakotoarison DA, Gressier B, Trotin F, Brunet C, Luyckx M, Dine T, Bailleul F, Cazin M, Cazin, J-C. (2002) Effects of Rosa canina fruit extract on neutrophil respiratory burst. Phytotherapy Research; 16(2):157 161 Kilicgun Hasan, Dehen Altiner (2009) In vitro Antioxidant Effect of Rosa canina in Different Antioxidant Test Systems. Phcog Res 2009;1:417-20 107
Influence of medium on growth and development of wild rose in vitro Parađiković N, Zeljković S, Vinković T, Mustapić-Karlić J, Kanižai G. (2009) Rast i razvoj kadife (Tageteserecta L.) pod utjecajem volumena supstrata i tretmana sa biostimulatorom. Zbornik radova 44.hrvatskog i 4. Međunarodnog simpozija agronoma Pati PK, Rath SP, Sharma M, Sood A, Ahuja PS. (2005) In vitro propagation of rose. Biotechnology advances, Elsevier, str. 163-176. Vinković T, Parađiković N, Teklić T, Štolfa I, Guberac V, Vujić D. (2009) Utjecaj biostimulatora na rast i razvoj rajčice (Lycopersicon esculentum Mill.) nakon presađivanja. Zbornik radova 44.hrvatskog i 4. Međunarodnog simpozija agronoma 108