Abstract: Light was found to be the most important factor affecting its seed germination. Under light conditions, soaking the seed for 30 min. in 50 ppm GA and 40°C hot water enhanced germination rate significantly. Under dark conditions, the seeds treated with KNO were the only ones germinating effectively. 3 The results suggest that Hypericum perforatum seeds exhibit both exogenous and endogenous dormancy. To over come former originating from a chemical inhibitor in exudate from capsule, washing with water, 40°C hot water and 50 ppm GA treatments; to over come later related with absence of light, 0.01 mol KNO3 treatment were recommended.
INTRODUCTION
The use of natural products with therapeutic properties is as ancient as human civilisation. The industrial revolution and the development of organic chemistry resulted in a preference for synthetic products for pharmacological treatment. But, there has been an increasing tendency in using of plant-originated raw matters in medicinal treatment due to some reasons such as synthetic products have some adverse effects on human health and are very expensive or plant-originated matters have multi functional effects in contrast to synthetic products (Rates, 2001). Today, about 25% of the drugs prescribed worldwide and 121 active compounds being in current use come from plant. Of the 252 drugs considered as basic and essential by the World Health Organisation (WHO), 11% are exclusively of plant origin and a significant numbers are synthetic drugs obtained from natural precursors. It is estimated that 60% of anti-tumor and anti-infectious drugs already on the market or under clinical trial are of natural origin (Shu, 1998). The vast majority of these cannot yet be synthesised economically and are still obtained from wild or cultivated plant (Hamburger and Hosstettmann, 1991).
One of plants used in drug production industry intensively is Hypericum perforatum. It, commonly known as St. John’s worth, is a medicinally important plant native to relatively dry temperature zones of Europe and North America (Deltito and Bayer, 1998). The species belonging to Hypericum genus have been used as traditional medicinal plants due to their wound-healing (Yazaki and Okuda, 1990), bactericide (Ishiguro et al., 1998), anti-inflammatory (Dias et al., 1998). Diuretic and sedative properties (Holz and Ostrowiski, 1987) for last two hundred years. Hypericum perforatum contains, namely naphthodianthrons and phloroglucinols, biologically active secondary metabolites belonging to at least ten different classes (Greeson et al., 2001). Hypericin and pseudo hypericin, one of components this plant has, exhibit antiviral (Meruelo et al., 1988; Lavie et al., 1989) properties. That is why, it is suggested that Hypericum perforatum has a potential in using of Acquired Immuno Deficiency Syndrome (AIDS) treatment (Schinazi et al., 1990). In the clinical studies, anti-tumor (Diwu, 1995; Agostinis et al., 1995) and anti-cancer (Takahashi et al., 1989; Hudson et al., 1991 and Lavie et al., 1995) properties of Hypericum perforatum were determined. Especially using of this unique plant as an antidepressant is very popular today.
There are about 300 Hypericum living in mild temperature zone of the world. Turkey is an important center for Hypericum. There are 69 Hypericum species in Turkey, 24 of which are endemic (Tokur, 1988). Hypericum perforatum found in Turkey commonly is worldwide plant economically due to various secondary metabolites it has. This is evidenced by the fact that the market for Hypericum perforatum has exceeded $210 million in USA and $570 million worldwide annually (Sirvent et al., 2002).
Germination is a critical stage in the life cycle of weeds and crop plants and often controls population dynamics, with major practical implications (Radosevich et al., 1997). But, generally germination rate of Hypericum perforatum is very low due to seed dormancy (Macchia et al., 1983). The dormancy is caused by a chemical inhibitor exudate from the capsule (Campbell, 1985) and absence of light has an negative effect on germination (Thompson and Whatley, 1984). This plant prefers to be propagated vegetatively (Tokur, 1988). Both low germination rate and difficulty in cultivating this plant using vegetatif plant parts make Hypericum perforatum production difficult (Mustyatse, 1983). Over the past twenty years, dormancy has been widely studied but the regulatory principles behind changes in several types of dormancy remain unclear (Rehman and Park, 2000). Nevertheless, plant growth regulators such as GA and IAA (Hilhorst and Karssen, 1992; Iglesias and Babiano, 1997); chemical matters like KNO3 (Kevseroğlu, 1993; Hartmann et al., 1997) and hot water treatments (Hermansen et al., 1999) has been recommended in breaking dormancy and to enhance germination. The objectives of this study were to determine the effect of light and exogenously applied GA, KNO3 and hot water on germination and to find an effective method for breaking seed dormancy of Hypericum perforatum L.
MATERIALS AND METHODS
In the study, the 6 month-old seeds obtained from Hypericum perforatum plants collected from the mountain pasture named Sisdağı in Trabzon province of Turkey were used. Before treating of different factors, seeds were bulked and washed with tap water to remove chemical inhibitor in exudate from capsule from seed surfaces (Campbell, 1985). Germination rates of the seeds exposed to different treatments were determined with enumerations made 20 days later.
Presoaking treatments used in the study were Different GA doses, hot water and 0.01 mol KNO3. Before placing in petri dishes, seeds were soaked in 50 and 100 ppm GA solution and 40 and 60°C hot water for 30 minutes. For KNO3 treatment, petri dishes were wetted with 0.01 mol KNO3 solution. To evaluate the effect of light on germination, the study was performed under both continuous illumination (1200 lux white light (Macchia et al., 1983) and darkness in growth chambers. Heat was fixed with 18°C (Campbell, 1985).
For each application 3×100 = 300 seeds were placed in petri dishes and germinating seeds were counted for 20 days after treatments. Obtained numbers were analysed according to design of randomised plots using MSTAT packet program. Differences among treatments were assessed according to least significant differences (LSD) test (Yurtsever, 1984).
RESULTS AND DISCUSSION
The germination rates of Hypericum perforatum seeds treated with different factors were shown in Table 1 as a group. All treatments applied in this study had a significant effect on germination rate (p<0.01).
According to the results of variance analysis, effect of light on germination rate was significant. As untreated control seeds gave second highest germination rate under light conditions with 60.67%, the lowest germination rates was obtained from control seeds under darkness (1.00%). These results are similar to that of Macchia et al. (1983), Thompson and Whatley (1984), Campbell (1985) and Tokur (1987) reporting that germination was very low in absence of light in Hypericum perforatum seeds. Also, germinating seeds under darkness formed etiolated seedling (Fig. 1a) but, that of germinating under light conditions were green and looks alive (Fig. 1b).
The effect of light on seed germination is various. Light has no effect on germination of many crops seeds economically important but, some of them need light to germinate at the highest level (Kacar, 1996). Likewise in a study conducted out to determine the effect of light on germination of seven different cacti species, Arechiga et al. (1997) obtained similar results.
As mean of light and dark conditions, among presoaking treatments, 40°C hot water and 50 ppm GA treatments gave the highest germination rates with 41.33 and 39.67%, respectively. The percentages were followed by 100 ppm GA and 0.01 mol KNO3 treatments found in the same statistical group (31.83 and 30.67%, respectively). The lowest germination rate was obtained from seeds treated with 60°C hot water. Hot water applications have enhanced germination of hard coated seeds by elevating water and O2 permeability of testa (Aydın and Uzun, 2001). Hermansen et al. (1999) reported that germination was enhanced by presoaking in 44, 49 and 54°C hot water for 5-40 minutes in carrot seeds. Hot water treatments also can have a detrimental influence on germination, which is special concern in high value seeds such as hybrid cabbage (Williams, 1980). In this study germination rate was very low in seeds presoaked in 60°C hot water. But, adverse effect of hot water on germination can be minimised by reducing the length of exposure to hot water (Smit and Davies, 1989).
Endogenus GAs are widely studied in relation to the breaking of dormancy in seeds of various plant species (Seiller, 1998). Imbibition stimulates GA secretion from embryo. Secreted GA increases synthesis of hydrolytic enzymes located under aleuron layer. Synthesised enzymes are transported to endosperm via scutulum and are used for decomposing of stored food to supply the energy required for germination (Cetin and Onay, 1994). In this study, GA increased germination rate significantly depending on used doses. Similar results were obtained from the studies carried on other species such as Haplopappus gracilis (Galli et al., 1975), Sesamum indicum (Kyauk et al., 1995), Rumex dentatus (Ali and Helal, 1996), Zea mays and Glycine max (Wang et al., 1996) and Opuntia tomentosa (Carrillo et al., 2003).
| Table 1: | The effects of light and some presoaking treatments on germination rates of Hypericum perforatum seeds |
LSD presoaking treatments: 6.497; LSD light×presoaking treatments: 9.188. CV:%13.07**There is no difference between means marked with the same letter in%1 probability level | |
| Fig. 1: | Germination of Hypericum perforatum seeds under light and dark conditions |
| Fig. 2: | Light×different presoaking treatments interaction |
Nitrates have been commonly used for breaking of dormancy in seeds requiring light for germination. Nitrates have increased seed germination by offsetting the light requirement on a large scale (Kacar, 1996). In a study whose main objective was to evaluate seed treatments for reducing or eliminating the light requirement of Lesquerella fendleri seeds, KNO3 was reported as an effective agent for reducing light requirement and enhancing germination (Puppala and Fowler, 2003). In this study, similarly, germination enhancing effect of KNO3 was more evident under darkness (Table 1).
Besides each treatment, an interaction (P<0.01) between treatments was determined as statistically significant (Fig. 2). In this respect, the highest germination rate was obtained from 50 ppm GA and 40°C hot water treatments (75. 33 and 72.67%, respectively) under light conditions. The numbers were followed by untreated control (60.67%) and 100 ppm GA treatment (59.33%) under light conditions. All treatments decreased germination rate under darkness statistically except for 0.01 mol KNO3 treatment when compared to that of light conditions.
According to the results of this study, H. perforatum seeds exhibit both exogenous and endogenous dormancy. Former originating from a chemical inhibitor in exudate from capsule could be eliminated 40°C hot water and 50 ppm GA treatments effectively. All treatments performed under light conditions increased germination rate when compared to that of darkness. But, 0.01 mol KNO3 was the only treatment causing significant increase for germination under darkness. The effect of KNO3 probably was related to its light requirement reducing property. The result suggest that there is an endogenous dormancy related to absence of light in Hypericum perforatum seeds except for exogenous dormancy.
In this study, effects of light and some presoaking treatments on germination rate of Hypericum perforatum were investigated. According to the results, light was the most significant factor affecting germination. Under light conditions, 50 ppm GA and 40°C hot water treatments increased germination significantly. Under darkness, the seeds treated with 0.01 mol KNO3 were the only ones germinating effectively. The results suggest that Hypericum perforatum seeds exhibit both exogenous and endogenous dormancy. To over come former originating from a chemical inhibitor in exudate from capsule, washing with water, 40°C hot water and 50 ppm GA treatments; to over come later related with absence of light, 0.01 mol KNO3 treatment were recommended. But, more studies will be needed about endogenous dormancy.