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African Journal of Biotechnology Vol. 9(39), pp. 6503-6508, 27 September, 2010 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 2010 Academic Journals Breaking seed dormancy in oil rose (Rosa damascena
Mill.) by microbial inoculation
Soner Kazaz1*, Sabri Erba 2 and Hasan Baydar2
1Department of Horticulture, Faculty of Agriculture, Suleyman Demirel University, Isparta 32260, Turkey. 2Department of Field Crops, Faculty of Agriculture, Suleyman Demirel University, Isparta 32260, Turkey. This study was carried out to determine the effects of microbial inoculation in breaking seed dormancy
and on the germination of Rosa damascena Mill. Seeds of R. damascena Mill. are the most used
scented rose species in rose oil production. The most important production centers around the world
are Turkey and Bulgaria. The seeds were subjected to 4 weeks of warm stratification at 25°C, followed
by 150 days of cold stratification at 4 ± 1°C. Before stratification, 4 different microbial fertilizers, EM•1®,
B:speel™, BioplinTM and Phosfert™ were inoculated to the seeds. In the study, the microbial
inoculation treatments significantly (p < 0.01) promoted the premature germination percentage during
cold stratification. During the stratification, the highest premature germination percentage was
obtained from the EM•1® (69.3%). The highest germination percentage in terms of cumulative
germination percentage was determined in EM•1® (100.0%), followed by Phosfert™ (84.0%) and B:
seepel™ (84.0%), whereas the lowest germination percentage was found in the control treatment
(69.3%). The EM•1® shortened the mean germination time by 1.7 days in comparison to the control. In
conclusion, it was observed that with microbial inoculation (particularly EM•1®) to oil rose seeds and a
stratification time of 150 days, dormancy was broken and germination highly improved.

Key words: Rosa damascena Mil , dormancy, germination, microbial inoculation, stratification.

INTRODUCTION
The genus Rosa has over 130 species (Cairns, 2001) Turkey, Bulgaria, India, Iran, Egypt, Morocco and Syria that are native to the Northern Hemisphere (Krüssmann, (Büttner, 2001), the most important production centers in 1981), and of these species, 25 are distributed in Turkey the world are Turkey and Bulgaria. R. damascena Mil ., a (Kutbay and Kilinc, 1996; Ercisli, 2004). The primary perennial shrub, produces pink flowers in May-June. Oil species used in rose oil production among rose species rose is a temperate zone plant and has wel adapted to are Rosa damascena Mil , Rosa gal ica Linn., Rosa climate zones, which receive abundant light and centifolia Linn. and Rosa moschata Herm. (Tucker and adequate rain, and do not experience negative climatic Maciarel o, 1988). Among these species, R. damascena factors such as drought, excessive rainfal and freezing is commonly used in oil production (Douglas, 1993). during the flowering period but in which dew occurs Although oil rose is cultivated in many countries such as during the early morning hours. The primary products that are obtained from oil rose and that are greatly demanded in cosmetics industries include rose oil, rose water, rose concrete and rose absolute (Kaur et al., 2007; Kazaz et al., 2009). Fruits, fruit flesh and seeds of R. damascena *Corresponding author. E-mail: skazaz@ziraat. sdu.edu.tr. Tel: contain ascorbic acid 332.0, 546.0 and 145.0 mg/100 g, +90 246 211 4656. Fax: +90 246 237 1693. respectively. Also R. damascena fruits can be used as food and food additive similarly as with dog rose fruits Abbreviations: ABA, Abscisic acid; PSB, phosphate
solubilizing bacteria; PGPR, plant growth promoting
(Rosa canina) (Kazaz et al., 2009). rhizobacteria; GP, germination percentage; MGT, mean
Rose seeds show both endogenous (morphological and/or physiological) and exogenous (physical and/or mechanical) dormancy (Gudin et al., 1990; Ueda, 2003). from the oil rose plantations in Isparta Province (Isparta, Turkey, Rose seeds are surrounded by a hard-coated pericarp, 37° 45' N latitude, 30° 33' E longitude and 997 m altitude) in October and the pericarp prevents water absorption and air diffu- 2008. Rose hips contain 2.35 seeds per hip on average. The sion of the seed and at the same time is a physical annual mean temperature, relative humidity, total annual precipi- tation, wind speed and sunshine duration per day in the area are barrier to embryo expansion (Ueda, 2003; Zlesak, 2007; 12.4°C, 55%, 524.4 mm, 2.4 m s-1 and 7.6 h, respectively Meyer, 2008). In addition, it was stated that high (Anonymous, 2003). With these climate characteristics, Isparta concentrations of abscisic acid (ABA) in the pericarp and features a semi-arid climatic characteristic (Ucar et al., 2009). testa of rose seeds was a major germination inhibitor in roses (Jackson, 1968; Cornforth et al., 1966; Bo et al., 1995; Hartmann et al., 2002). It was reported that the Experimental site
amount of ABA in a rose seed was 10- to 1000-fold The research was conducted in a plastic covered greenhouse higher than those in other plants (Ueda, 2003). Due to located at the Agricultural Research and Application Center of the above-mentioned reasons, the germination of rose Agricultural Faculty at Süleyman Demirel University (latitude 37° 50' seeds is general y difficult. Prolonged dormancy delays N, longitude 30° 32' E, altitude 1019 m). germination and reduces germination percentage. This is a serious problem particularly in rose breeding and seed propagation (Yambe and Takeno, 1992; Bo et al., 1995; Seed preparation and determination of moisture content and
1000 seeds weight
The degree of dormancy varies by the time and After the seeds had been manual y extracted from hips, they were temperature required to overcome dormancy as wel as cleaned in water and the unwanted materials were removed. Later, by germplasm, maturity at hip col ection, time of seed the seeds were soaked in water for 24 h and then the floating seeds extraction, temperatures during seed development and were discarded and the seeds that sunk in water were used in the temperature and duration of stratification (Semeniuk and treatment as they were assumed to be mature and viable (Zhou et Stewart, 1962; Gudin et al., 1990). One of the most com- al., 2009). After the seeds had been dried in the open air for 3 days, monly used methods to break dormancy and stimulate they were kept in polyethylene bags at room temperature (20 - 24°C) until the beginning of the treatments. Seed moisture content germination in rose seeds is stratification (Zlesak, 2007). (four replicates of 100 seeds) was determined at 103°C for 17 h Various methods, such as gibberel ic acid (Hosafci et al., and 1000 seeds weight was determined based on 8 replications of 2005), hot water treatment (Younis et al., 2007), scarifi- cation with sulphuric acid (Bhanuprakash et al., 2004) and macerating enzymes (Yambe and Takeno, 1992), have also been tried besides stratification. Even though Microbial treatments and warm plus cold stratification
these methods are used alone or as a combination, it has been reported that the germination percentages in some Some 4 different microbial fertilizers (EM•1® EM Agriton and Kina- gro Agriculture Inc, Turkey), B: speel™ (Bioglobal Inc. Turkey), rose species are stil low. It was reported that the Bioplin™ (Bioglobal Inc, Turkey) and Phosfert™ (Bioglobal Inc, germination percentages ranged from 0 to 10% in the 1st Turkey) were used in the study. EM•1® primarily contains 3 types of year and from 24.7 to 73.7% in the 2nd year (Hosafci et microorganisms, namely phototrophic bacteria (Rhodopseudomonas al., 2005). Bel etti et al. (2003) reported that they ranged palustris), lactic acid bacteria (Lactobacil us plantarum, Lactobacil us from 0.5 to 50.3% and that this percentage was 18.8% in casei, Lactobacil us fermentum and Lactobacil us delbruecki ) and yeasts (Saccharomyces cerevisiae). B:seepel™ is a bioorganic R. canina L. according to Alp et al. (2009), while the ger- seed dresser and contains a mixture of microorganisms (1x107 mination percentages were 13.8 and 13.5% in Rosa cfu/g) fixing nitrogen in dormant form, a mixture of phosphate pulverulenta Bieb. and Rosa dumalis Bechst., respec- solubilizing bacteria (PSB) (1x107 cfu/g), plant growth promoting tively (Alp et al., 2009). In Rosa bracteata Wendl, they rhizobacteria (PGPR) and metabolic extracts of different microbes. ranged from 1.8 to 41.5% according to Anderson and Bioplin™ contains efficient rhizosphere inhabiting, nitrogen fixing and plant growth promoter producing strains of Azotobacter (Azotobacter chroococcum and Azotobacter vinelandi 1 x 107 One of the methods used to break dormancy in seeds cfu/g). Phosfert™ contains plurality of strains of Azotobacter (A. and promote germination percentage is microbial chroococcum, A. vinelandi , Bacil us polymyxa 1 x 107 cfu/g). inoculation to seeds or germination medium. It was Firstly, the seeds were left in water for 24 h and then they were reported that microorganisms macerated the hard-coated left in Bioplin™ (15 ml/l), Phosfert™ (15 ml /l) and Phos- seed pericarp and facilitated germination (Morpeth and fert™+Bioplin™ (1:1, v/v) solution for 15 min and in EM•1® solution Hal , 2000). The objective of this study is to determine the (300 ml /l) for 20 min. In the B:seepel™ treatment, B:speel™ (20 g/kg seed) was sprinkled over the seeds, and the seeds were effects of microbial inoculation in breaking seed dorman- covered completely with B:speel™. On the other hand, no microbial cy and on the germination of R. damascena Mil . seeds. fertilizer treatments were performed on the seeds in the control Stratification was applied to the seeds treated with microbial MATERIALS AND METHODS
fertilizer and to the seeds of the control group. Sphagnum moss was used as the stratification medium. Those seeds that were Seed origin and seed collection
mixed with moistened sphagnum moss (1 part of seed and 4 parts of sphagnum moss, v/v) were subjected first to 4 weeks of warm The mature hips of the species R. damascena Mil . were col ected stratification at 25°C and then to 150 days of cold stratification in Table 1. Effects of microbial inoculations on seed germination percentage (%) and mean germination time (day).
Treatment
Premature
Greenhouse
Cumulative
Mean germination
germination1 (%)
germination2 (%)
germination3 (%)
time (days)
1Germination during stratification; 2germination in greenhouse (seeds without premature germination); 3premature germination plus **Mean values in the same column fol owed by the same letter are not significantly different at the 0.01 level according to the Duncan’s test. refrigerator at 4 ± 1°C in polyethylene bags. In order to keep spha- greenhouse-germinated seeds, and the MGT were analyzed using gnum moss moist in the stratification medium and for aeration, the SAS (1998) statistical analysis program. The germination percen- polyethylene bags were opened once a week during the stratifi- tages were transformed into arcsine before analysis. After cation period, and water was added as needed. evaluation, data were back transformed and original data presen- ted. The mean values were compared by Duncan’s multiple range Germination experiment
At the end of stratification, premature germination took place in al treatments, except for Bioplin™. The number of prematurely germi- nated seeds in each treatment was recorded, and the germination percentages of these seeds were further analyzed in order to Germination percentages
determine the difference between the treatments. The prematurely germinated seeds were not sown in the germination medium in the In this study, moisture content of seeds was 11.15%, and greenhouse, and only those seeds that did not germinate at the end weight of 1000 seeds was 20.9 g. Microbial inoculation of duration of stratification were sown. The seeds treated with warm plus cold stratification were sown in peat-containing vials in the treatments significantly (p < 0.01) stimulated premature plastic covered greenhouse on May 28, 2009. The misting irrigation germination during cold stratification. At the end of this system was used with adequate moisture both in the greenhouse period, premature germination was observed in al treat- and in the germination medium after the sowing of seeds. ments, except for the Bioplin™. The highest premature Germination tests were carried out in greenhouse at 25°C day/15°C germination percentage was determined in the EM•1® night temperature and a relative humidity of 70%. A seed was (69.3%), fol owed by B:seepel™ (52.0%) and Phosfert™ considered to have germinated when the cotyledons had emerged above the soil surface, and it was recorded for up to 30 days. (44.0%). However, premature germination was 13.3% in Germinated seeds were counted and removed every 24 h for 30 the seeds treated only with warm plus cold stratification days. Final germination percentage was calculated when no further germination took place for several days. The germination percen- The germination percentages of seeds sown in the tage (GP) was calculated for each experimental unit. Mean greenhouse after cold stratification are presented in germination time (MGT) was calculated using Equation (1) (Chuanren Table 1. Statistical y significant differences were determi- ned between the germination percentages of the treatments (p < 0.01). Among the treatments, the highest germination percentage was obtained in the EM•1® Where, n is the number of seeds that germinated between scoring (100.0%), whereas the other treatments were included in intervals; d the incubation period in days at that point in time and N the total number of seeds that germinated in the treatment. When the germination percentages of prematurely germinated seeds at the end of the duration of stratifi-
Experimental design and data analysis
cation and of greenhouse-germinated seeds were considered together (cumulative germination percentage), A completely randomized plot design of 3 repetitions was used, and microbial inoculation treatments statistical y significantly each replication consisted of 25 seeds. The percentage of prema- affected cumulative germination percentage. Al seeds turely germinated seeds during cold stratification in the experiment, germinated with the EM•1®. Furthermore, Phosfert™ and the germination percentage of those seeds that were not B:seepel™, with their germination per-centage of 84%, germinated at the end of the duration of cold stratification and sown in the greenhouse immediately afterwards, the cumulative germina- were included in the same group with EM•1®. 66.7% tion percentage of both prematurely germinated seeds and the germination occurred in the seeds (control) which were
Figure 1. Effects of microbial inoculations on seed germination (%).
only stratified without any microbial inoculation treatments. stratification to obtain maximum germination percentages (Steward and Semeniuk, 1965). Moreover, it was reported that a stratification duration longer than 150 Mean germination time
days was needed to remove embryo dormancy of oil rose seeds and that the germination percentage was over No statistical difference in mean germination time was 80% through soaking seeds in 70 and 80% sulphuric acid found between microbial inoculation treatments and the for 10 min fol owed by 150 to 180 days of stratifi-cation control treatment. Nevertheless, although no statistical (Hajian and Khosh-Khui, 2000). Higher germi-nation difference was found between treatments, the mean percentages were obtained in this study. The higher germination time of the EM•1® (7.2 days) was 1.7 days premature germination percentage of oil rose seeds in al shorter than that of the control (Table 1). microbial inoculation treatments except for Bioplin™ during stratification than the control treatment might be due to an increase in the number of microorganisms in DISCUSSION
the seed pericarp during stratification and might be because these microorganisms macerated the hard and This study showed that the germination percentage of oil thick seed pericarp, thereby facilitating germination. A rose seeds was significantly affected by microbial similar case was reported by Morpeth et al. (1997) and inoculation. During 150 days of cold stratification fol o- wing 4 weeks of warm stratification, premature germi- In this study, microbial inoculation treatments signifi- nation was observed in seeds in al treatments, except for cantly increased germination percentage in comparison the Bioplin™. This indicates that the stratification duration to the control. The results of the present study are also of 150 days might be adequate to break dormancy of the supported by the findings of Morpeth and Hal (2000) in seeds of the species R. damascena. The most common Rosa corymbifera (95%) and of Bel etti et al. (2003) in R. treatment to break dormancy of rose seeds is cold canina (50.25%) that microbial inoculation to the seeds stratification (Zlesak, 2007; Zhou et al., 2009), and the degree of dormancy varies by species and duration of Among the treatments, the highest germination percen- stratification (Stewart and Semeniuk, 1965). For instance, tages were obtained from the EM•1®, fol owed by the the species Rosa multiflora and Rosa setigera need 30 Phosfert™ and B: speel™ (Figure 1). In both the prema- days of cold stratification; the species Rosa wichuraiana turely germinated seeds during stratification and those needs 45 days of cold stratification; and R. setigera seeds that did not germinate during stratification but 'Serena' and Rosa x reverse need 90 days of cold germinated in the greenhouse immediately afterwards,
Figure 2. Effects of microbial inoculation on mean germination time (days).
the lowest cumulative germination percentage was nated with the EM•1®. The observation of a high rate of obtained from the control treatment (66.7%). Although premature germination (69.3%) of the R. damascena there was no statistical y significant difference between seeds during stratification with the EM•1® indicates that the Phosfert™, B: speel™ and the control (which might the time required for stratification in this species might be be because the EM•1® showed a very high germination further reduced with the EM•1®. The inoculation of micro- percentage), both treatments showed a 20.6% higher organisms to the seeds during preliminary treatment and germination percentage than that of the control treatment the development of microorganisms immediately after- in terms of cumulative germination percentages. It might wards facilitated the germination of seeds. The study also be stated that this percentage is quite high in commercial showed that 150 days of cold stratification (4 ± 1°C) fol owing 4 weeks of warm stratification (25oC) might be The effect of treatments on the mean germination time enough to break dormancy. How long it takes for dorma- of oil rose seeds was statistical y insignificant. However, ncy of the species R. damascena to be broken wil be despite the statistical y insignificant difference among clarified with further studies that we wil be later them, the mean germination times in EM•1® (7.2 days) conducted on EM•1® and other microbial fertilizers with and B: speel™ (7.3 days) were 1.7 and 1.6 days shorter than that of the control, respectively (Figure 2). Bel etti et al. (2003) reported that different doses of compost activator treatments in R. canina further shortened the REFERENCES
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