Effects of Ginkgo Biloba L. Extract on the Seed Germination, Seedling Growth and Leaf Anatomy of Barley under Saline Conditions

Effects of Ginkgo biloba L. extract on the seed germination, seedling growth and leaf anatomy of barley under saline conditions were studied. In parallel with concentration rise, salt stress inhibited the seed germination and seedling growth of barley. The inhibitive effect of salt on the germination and coleoptile percentage was alleviated in varying degrees, and dramatically, by Ginkgo biloba application. However, it became ineffective in alleviating of salt inhibition on the radicle, coleoptile length, radicle number and fresh weight of barley seedlings. On the other hand, it was observed that Ginkgo biloba extract affected in different degrees the various parameters of leaf anatomy of barley seedlings, and this difference was statistically important. Introduction Nearly 20% of the world’s cultivated area and half of the world’s irrigated lands are affected by salinity (Zhu 2001). The salt-affected soils contain excess salts which affect plants by decreasing the osmotic potential of the soil solution (osmotic stress), interfering with normal nutrient uptake, inducing ionic toxicity, and associating nutrient imbalances (An et al. 2003). Processes such as seed germination, seedling growth and vigour, vegetative growth, flowering and fruit set are adversely affected by high salt concentration, ultimately causing diminished economic yield and also quality of production (Sairam and Tyagi 2004). In addition, it is evident that there are big changes in leaf morphology and anatomy of the plants growing in saline soils (Çavuşoğlu et al. 2008). Ginkgo biloba L. (GB) is probably the oldest known tree species, dating back to 300 million years and it is often called the “living fosil” (Mariassyova 2006). More than 20 Ginkgo variations exist that differ in tree habitat and in shape, colour and size of leaves, the latter being the raw material for the pharmaceutical industry (Ellain-Wojtaszek et al. 2002). Especially the leaves of GB contain compounds possessing an antioxidant character (Zahradnikova et al. 2007). There have been isolated three main compounds from GB with an antioxidant activity-kaempferol, quercetin and isoharmnetin (Spence and Jane 1999). The antioxidant activity of a Ginkgo extract is determined mainly by flavonoids, which scavenge and destroy free radicals and the reactive forms of oxygen (Ellain-Wojtaszek et al. 2002). GB leaf extract is the most widely sold phytomedicine in Europe, where it is used to treat the symptoms of early-stage Alzheimer’s disease, vascular dementia, premenstrual problems, peripheral claudication, altitude sickness and tinnitus of vascular origin (Sierpina et al. 2003). Although there are many clinical reports on GB, unfortunately, the protective mechanisms of GB on salt stress in plants are stil poorly understood. The purpose of this study is to observe the influences of GB extract in reducing the inhibitive effects of salt stress on the seed germination, seedling growth and leaf anatomy of barley. *Author for correspondence: . 118 ÇAVUŞOĞLU AND KARAFERYELİ Materials and Methods Barley (Hordeum vulgare L. cv. Bülbül 89) seeds were used. Salt (NaCl) concentrations used were 0.0, 0.25, 0.275 and 0.30 M. Ginkgo biloba (GB) concentration used in the experiments was 0.5 ppm. Germination experiments were carried out at a constant temperature (20°C), in the dark in an incubator. Barley seeds in adequate amount were pretreated in the beakers containing sufficient amount of distilled water (control, C) or aqueous solution of GB for 24 h at room temperature. At the end of this pretreatment, the solutions were filtered immediately and the seeds were dried in vacuum (Braun and Khan 1976). Twenty five seeds from every application were arranged into Petri dishes (10 cm diam.) lined by 2 sheets of Whatman No. 1 filter paper moistened with 7 ml of the salt solution. After sowing, Petri dishes were placed into an incubator for germination for 7 days. It was assumed that the radicle should be 10 mm long for germination. At the end of the 7th day, after determination of the final germination percentages, the coleoptile emergence percentages and radicle numbers were also recorded, and in addition, the fresh weights in mg/seedling were determined. All experiments were repeated four times. The seedlings from the seeds germinated in the incubator at 20°C for 7 days were transferred to pots with perlite including NaCl solutions prepared with Hoagland recipe and were grown in a growth chamber for 20 days. Anatomical sections were taken from the second leaf of 20-day-old seedlings by a microtome, in 6-7 μm thickness. They were examinated under a binocular light microscope (Olympus CX41) at 100 magnification. Stomata and epidermal cells in a 1-mm unit area were counted to determine the stomatal index. These counts were made both in the lower and upper surfaces of each leaf 10 times as 3 replicates and the averages were calculated. After the determination of the number of stomata and epidermal cells in the leaf unit area, the stomatal index was estimated according to Meidner and Mansfield’s (1968) method. Stomata width and length, epidermal cell width and length, leaf thickness and distance between vascular bundles were also determined. Statistical evaluation concerning all parameters was realized by using SPSS program according to DMRT. Results and Discussion Results showed that GB application increased the germination and coleoptile percentage while it decreased the coleoptile length and fresh weight. In addition, it statistically showed the same values as the C regarding the radicle lenght and radicle number (Table 1). Çavuşoğlu et al. (2010) reported that GB partly reduced the germination percentage of Vicia faba seeds in distilled water medium. This result was not consistent with the present findings. Howewer, they also observed that GB did not show a meaningful effect on the radicle length of V. faba seedlings and this is consistent with the present findings. It can be said that GB can show different effects on seed germination and seedling growth depending on the plant species and the concentrations used. Salt, in the paralellism of concentration increase, increased its inhibitive effect on all the examined growth parameters. For example, while C seeds germinated in distilled water medium showed 94 % germination on the 7th day, this value became 35, 24 and 18%, respectively in 0.25, 0.275 and 0.30 M salinity (Table 1). On the other hand, GB application markedly alleviated the inhibitive effect of salt stress on the seed germination. The seeds pretreated with GB demonstrated 77, 65 and 56% germination in the above mentioned salt levels. GB also continued its success on the coleoptile percentage. However, it was ineffective in alleviating salt inhibition on the radicle length, coleoptile length, radicle number and fresh weight of barley seedlings (Table 1). It is possible that GB may be effective in alleviating the inhibitive effect of salt on the seed germination by increasing nucleic acid and protein synthesis, by stimulating mitotic activity of EFFECTS OF GINKGO BILOBA EXTRACT ON BARLEY SEED GERMINATION 119 embryo, by providing stabilization of cell membranes or by raising antioxidant enzyme activities (Ellain-Wojtaszek et al. 2002, Zahradnikova et al. 2007). Table 1. Various growth parameters of the seedlings from barley seeds germinated in saline conditions for 7 days.