EFFECT OF WATER STRESS BY POLYETHYLENE GLYCOL 6000 AND SODIUM CHLORIDE ON SEED GERMINATION AND SEEDLING GROWTH OF CASSIA ANGUSTIFOLIA

EFFECT OF WATER STRESS BY POLYETHYLENE GLYCOL 6000 AND SODIUM CHLORIDE ON SEED GERMINATION AND SEEDLING GROWTH OF CASSIA ANGUSTIFOLIA S. M. Shitole and K. N. Dhumal Department of Botany, University of Pune, Pune- 411007, Maharashtra, India

ABSTRACT:The impact of salt stress and water stress on seed germination, root and shoot length, fresh and dry biomass, vigour index was investigated in Senna (Cassia angustifolia) at germination stage (7DAS). Salinity stress was induced by using NaCl and drought stress by using PEG-6000. Different concentration of NaCl (0.1 to 100 mM) and PEG-6000(-0.1 bars to -2.0 bars) were used for seed treatment. The objective of the present investigation was to study the comparatively impact of NaCl salinity stress and water stress on seed germination, seedling growth, fresh and dry weight in Senna.

INTRODUCTION: Seed germination is one of the most important phases in the life cycle of plant and is highly responsive to existing environment ¹. The abiotic factors such as salt and drought stresses are the two of the most important abiotic stresses that limit seed germination and seedling growth ^{2, 3, 4, 5}. Abiotic stress conditions negatively influence the survival of plants, biomass accumulation and yield ⁶. Osmotic adjustment plays an important role in sustaining growth under water deficit conditions. The response of plants differs significantly at various organizational levels, depending upon intensity and duration of stress as well as plant species and its stage of development ⁷. Both drought and salt stress is harmful during early stages of seed germination and seedling growth. With increasing realization of health hazards and toxicity associated with the indiscriminate use of synthetic drugs and antibiotics, more and more people are interested in the use of plants and plant based drugs being revived through out the world. So exploitation of medicinal plants has become more and more popular. For the past several years, several scales of physiology have been applied to study responses to salt stress tolerance mechanisms and methods to overcome salt stress in field crops ⁸. It seems necessary to do research related to the correlation between medicinal plants and salt stress for the increasing need of medicinal plants. In order to meet the ever increasing demand of medicinal plants, for the indigenous systems of medicine as well as for the pharmaceutical industry, some medicinal plants need to be cultivated commercially, but the soil salinity and other forms of pollutions pose serious threats to plant production ⁹. So it seems valuable, to test the important medicinal plants for their salt tolerance capacity. Cassia angustifolia (Family: Caesalpiniaceae), popularly known as Senna, is a valuable plant drug in Ayurvedic and Modern System of Medicine. The primary medicinal components of Senna is sennoside A and B, are the two anthroquinone glycosides that are responsible for purgative action of Senna ¹⁰ and is used to cure a large number of intestinal diseases. Therefore, the objective of this research was to examine the influence of different concentrations of NaCl and PEG 6000 on the seed germination, root and shoot length, fresh and dry weight of Cassia angustifolia. MATERIALS AND METHODS: Authentic seeds of Cassia angustifolia Vahl were obtained from National Research Centre for Medicinal and Aromatic Plants, Boriavi, Gujarat. Seed germination bioassay was carried out during the month of July- August (2008-2009). The seeds were surface sterilized with 0.1 % HgCl₂ for five minutes with intermittent shaking; and thoroughly rinsed with distilled water. These seeds were put in 9 cm diameter sterilized petri plate containing single layer of seed germination paper. Salinity stress was induced by using NaCl and drought stress by using PEG-6000. Different concentration of NaCl (5 to 100 mM) and PEG-6000 (-0.25 bars to -2.0 bars) were used for seed treatment. The solution of different concentrations of Polyethylene Glycol (PEG 6000) was prepared by the method of Michel and Kaufmann ¹¹. Five ml solution of NaCl (5, 10, 25, 50 and 100 mM) and PEG 6000 (-0.25, -0.50, -1, -1.5, -2) bar were added in different petri plates. The control contained five ml of sterile distilled water. Each treatment was maintained in triplicate along with control. About 20 seeds were kept in each petri plate for germination. The germination studies were carried out in seed germination chamber at 30 ± 2^oC temperature, 94 ± 2 relative humidity and uniform light conditions. The germination percentage, the root and shoot length (cm), fresh and dry weight (gm) were measured after 7^th day of germination in all the germinated seedlings. The seedling dry weight was determined after drying at 80^oC for 24 h. The average values were recorded. For seed germination experiment the control is always DW. Vigour Index was calculated by using formula, Vigor Index (VI) = Germination percentage × (Root length + Shoot Length) Statistical Analysis: The data was presented as arithmetic means of three replicates ±standard deviation. The significance of the mean differences was explored through one-way-ANOVA statistics followed by DMRT (Duncan’s multiple range test) at p=0.05 as a post hoc test. SPSS for Windows ver. 11.5 and Microsoft Excel 2003 were used to carry out statistical analyses and graphical data presentations. RESULTS AND DISCUSSION: Effect of NaCl and PEG 6000 on seed germination and seedling growth: Decreasing osmotic potential (increasing NaCl and PEG 6000 concentration) caused reduction in seed germination percentage (%). The reduction in seed germination was proportional to the increasing concentration of NaCl and PEG 6000. Maximum retardation was noted at highest NaCl concentration (100mM) and PEG 6000 (-2 bars). There was absolute (100%) inhibition of germination above these concentrations. Similar results were observed by ¹² Woodell ¹³ (1985); Gupta ¹⁴ Ungar ¹⁵ (1993), Safarnejad, ¹⁶ (2008). This was attributed to the effect that seeds seemingly develop an osmotically enforced inhibition by NaCl and drought stress. This may be an adaptive strategy of seeds to prevent germination under stressful environment for ensuring proper establishment of the seedlings. Not only the seed germination but seedling growth was also obstructed by chloride and water stress. These results indicated that the radicle and plumule growth was adversely affected. The shoot and root length of senna was negatively influenced by NaCl and PEG. This ultimately affected the fresh and dry weight of the seedlings. Similar results were observed by Pratap and Sharma ⁶ (2010), Kaydan and Yagmur, ⁵ (2008). It was claimed that decrease in the water potential gradient between seeds and their surrounding media adversely affect the seed germination and subsequent events in seedling growth and development. Water uptake leads to the activation of the metabolic processes as the dormancy of seed is broken ¹⁷. High concentration of NaCl and PEG might have hampered the process of water uptake by seeds and thereby inhibited the process of seed germination ¹⁸. Water and salinity stress induced by PEG and NaCl caused reduction in seed germination and also delayed germination time ⁵. Similar results for decreased seed germination and seedling growth due to NaCl and PEG ^{19, 20}. PEG and NaCl decreased germination in pea, but effect of PEG on germination is more than NaCl ²¹. Seed germination and seedling growth in legumes decreased under drought (PEG) treatment ²². Drought stress cause reduction of seedlings growth in beet ²³. Germination percent, radical and plumule length, fresh and dry weight of pea seedling decreased by increase of drought stress (PEG) ²¹. Drought stress caused reduction of germination rate and seedlings growth in okra plant ²⁴. Root length is an important trait against drought stress in plant varieties; in general, variety with longer root growth has resistant ability for drought ^{25, 26}. Seed germination, shoot and root length was decrease due to PEG in lentil ²⁷. NaCl stress decreased root and shoot length, dry matter production of Glycine max seedlings ²⁸.  Shoot and root length, fresh and dry weight of the seedlings were also reduced in both salinity as well as drought stress which may be due to the metabolic disorders induced by stress and generation of Reactive Oxygen Species (ROS). There is a significant reduction in early seedling growth at higher concentrations of NaCl and PEG-6000 due to their toxic effect at high concentration on the seeds (table 1). TABLE 1: EFFECT OF NaCl SALINITY STRESS ON SEED GERMINATION AND SEEDLING GROWTH OF SENNA

Both seed germination and seedling growth was reduced due to NaCl and PEG induced osmotic stress. However, PEG induced osmotic stress caused more growth inhibition as compared to NaCl induced osmotic stress ^{18, 29, 30}. Drought stress induced by using PEG-6000 was most harmful to plant as compared to NaCl stress. Water deficient conditions created by PEG decreased seed germination and seedling growth with higher negative effects. The explanation of the higher inhibitory effects of PEG than NaCl lies in ion or solute entry into the seed ²⁰. Especially, the accumulation of Na⁺ by the imbibing seed embryo functions to promote a water potential gradient between the embryo and substrate, and maintain water uptake during seed germination ^{19, 31} (table 2). TABLE 2: EFFECT OF PEG-6000 ON SEED GERMINATION AND SEEDLING GROWTH OF SENNA

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