Botany Seed germination of the halophyte Anabasis setifera (Amaranthaceae) from Saudi Arabia. Journal: Botany Manuscript ID cjb-2018-0053.R1 Manuscript Type: Article Date Submitted by the Author: 19-May-2018 Complete List of Authors: Basahi, Mohammed; Shaqra University College of Science and Arts Sajir, biology; Anabasis setifera,Draft halophyte, Temperature, Germination, seed germination Keyword: recovery Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/botany-pubs Page 1 of 27 Botany Seed germination of the halophyte Anabasis setifera (Amaranthaceae) from Saudi Arabia. Mohammed A Basahi College of Science and Arts Sajir Shaqra University P.O. Box 33, Shaqra 11961 Saudi Arabia [email protected] Draft00966582223689 1 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 2 of 27 Abstract The main objective of this study was to determine the effects of temperature, light/darkness, and salinity (NaCl) on seed germination of Anabasis setifera Moq. and the effects of alleviating salinity stress using distilled water. One-hundred percent of seeds completed germination at 15/5, 20/10, and 20°C, and a higher percentage of seeds completed germinationin light than in the dark at 20/10 and 25/15°C. The percentage of seeds that completed the germination decreased as salinity increased from 0 to 700 mM NaCl. Seeds that did not complete germination in the 800 or 700 mM NaCl solutions completed its germinationDraft after being transferred to distilled water, with a recovery rate of 94.5% and 75.5%, respectively, at 25/15°C. The inhibitory effect of NaCl on the completion of germination in this species probably occurs via an osmotic effect. Key Words: Anabasis setifera, halophyte, temperature, germination, seed germination recovery 2 https://mc06.manuscriptcentral.com/botany-pubs Page 3 of 27 Botany Introduction Germination and establishment are essential in the life cycle of halophytes (Ungar 1978). Different species of halophytes, such as Haloxylon recurvum Bunge ex Boiss, Atriplex triangularis Willd., Salicornia europaea L., Salicornia bigelovii Torr., Salicornia stricta Durmort., Salicornia rubra Nelson, Zygophyllum simplex L., Triglochin maritima L., Salicornia pacifica Standley, Arthrocnemum indicum Willd., and Diplachne fusca L. germinate in response to different stimuli (Langlois 1966; Ungar 1967; Rivers and Weber 1971; Chapman 1974; Khan and Ungar 1984, 1996b, 1999; PhilipupillaiDraft and Ungar 1984; Khan and Weber 1986; Myers and Morgan 1989; Khan and Gul 1998; Khan et al. 2000). These stimuli include environmental variables, such as salinity and temperature. For example, El-Keblawy et al. (2016a) investigated how temperature affected germination of Anabasis setifera Moq. seeds that were collected from the United Arab Emirates (UAE) and Egypt. Halophytes germinate in saline environments during the rainy season when the salinity of the surface soil layers decreases (Chapman 1960; Waisel and Ovadia 1972; Ungar 1978, 1982, 1987b; Ismail 1990). However, most plants, including halophytes, have a higher germination rate in distilled water, and this decreases as salinity increases (Rozema 1975; Ungar 1978; El- Sharkawi and Springuel 1979; Woodell 1985). El-Sharkawi and Springuethel (1979) found that the final percentage of germination in many species is affected by salinity at varying temperatures. Many environmental factors control halophyte germination in nature, including 3 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 4 of 27 light, temperature, and salinity (Badger and Ungar 1989; Gutterman 1993; Ungar 1995; Huang and Gutterman 1998; Khan 2003). Ungar (1982) showed that salinity and temperature were the most important factors, since they determine the time taken for halophyte seeds to complete germination. Badger and Ungar (1988) stated that for plants to successfully establish in a saline environment, the duration of germination is essential. Additionally, seedlings are more vulnerable to physical environmental changes than other life cycle stages. Therefore, the most important factor for halophytes to successfully establish in inland saline habitats is the timing of germination. Ungar (1987a) argued that seasonal variation in soil salinity may result in the extinction of entire plant populations. The halophyte Anabasis setifera (Amaranthaceae)Draft is broadly distributed within the coastal saline environments of Saudi Arabia, Afghanistan, Iran, India, and Pakistan. The species is often found alongside other halophytes, such as Suaeda vermiculata Forssk., Prosopis farcta (Banks & Sol.) J. F. Macbr., Suaeda aegyptiaca (Hasselq.) Zohary, and Atriplex leucoclada Boiss. (Mandaville 1990; Migahid 1996; AL-Turki 1997; Collenette 1998, 1999; Chaudary 1999). El-Keblawy et al. (2016a) reported that many germination studies have been conducted on Anabasis setifera seeds that originate from the UAE and Egypt, but none of them have used seeds from Saudi Arabia, where conditions are different. This study examined how A. setifera seeds from the saline marshes of Tarut Island on the Gulf coast of Saudi Arabia, respond to completion of seeds germination. We investigated seed germination responses to: (a) a broad range of constant and variable temperatures at a 4 https://mc06.manuscriptcentral.com/botany-pubs Page 5 of 27 Botany photoperiod of 12 hours daily; (b) uninterrupted light (20/10 °C) or constant dark (25/15 °C); and (c) increasing salinity stress. Materials and methods Collection of seeds A. setifera seeds were collected from Tarut Island (26°34'18.58" N, 50°03'40.57" E) on 22 December, 2015 (Figure 1). The seeds were air-dried, cleaned, and used immediately in germination assays. Effects of light/dark and temperature on germination Tests of seed germination were carriedDraft out using 9-cm Petri dishes containing two filter paper layers (Whatman no. 1) moistened with about 10 ml of distilled water. Five replicate Petri dishes with 20 seeds each were used for each treatment. The Petri dishes were distributed randomly in temperature-controlled incubators, and their positions were changed daily. The first emergence of the radicle from the seed was defined as germination (Côme 1982; Redondo et al. 2004). Observations were made daily for about 1 month and newly germinated seeds were removed from the Petri dishes. Seeds were incubated at one of five variable temperature regimes (35/25, 30/20, 25/15, 20/10 and 15/5 °C), or four constant temperatures (40, 30, 20, and 10 °C) and a daily photoperiod of 12 h light: 12 h dark. The variable temperature regimes simulated the likely diurnal changes in temperatures in the natural habitat. The final percentage of germinated seeds was calculated as: (1) G (%) = (A/B) × 100 where, A represents the total number of germinated seeds in 30 days, and B represents the total number of seeds tested (100 seeds) (Li and Shi 2010; Wang et al. 2013). 5 https://mc06.manuscriptcentral.com/botany-pubs Botany Page 6 of 27 The rate of germination (50% = t50) was calculated as:(2) GSI = G1/N1 + G2/N2 + ... Gn/Nn where, G1/G2/Gn represent the number of seeds that germinated, and N1/N2/Nn represent the duration of the experiment (Maguire 1962). To test the effects of light/dark on germination, seeds were incubated under continuous light or dark at 20/10 and 25/15°C, respectively. Germination of seeds incubated in the light was monitored daily, while those incubated in the dark were observed after 15 days (Qu et al. 2008). Effects of salinity on seed germination and germination recovery A total of nine salinity treatments were used: 0, 100, 200, 300, 400, 500, 600, 700, and 800 mM of NaCl. Seeds were germinated in 9-cmDraft Petri dishes with two layers of Whatman No. 1 filter papers, then incubated at 12:12 h light:dark at temperatures of 25:15 °C for about 1 month. Each treatment had five replicates with 20 seeds each. The Petri dishes were watered with 7 ml of their respective salinity solutions, and sealed using Nescofilm to prevent evaporation. The solutions were replaced every 7 days. Germinated seeds were counted daily for a period of 30 days and seedlings were removed from the petri dishes. Seeds that failed to germinate within the 30-day period were transferred into distilled water and given an additional incubation period of 15 days in 12:12 h light :dark at temperatures of 25/15 °C . After the 15-day period of recovery, the non- germinated seeds were tested for viability using 2,3,5-triphenyl tetrazolium chloride (TTC) solution, as recommended by the International Seed Testing Association (1999). The seeds were soaked in a solution of 1% TTC for 4 days in a glass vial in the dark at a temperature of 25 °C. The dehydrogenase enzymes in the living tissue reduces the colourless solution of tetrazolium chloride to insoluble red formazan, making the living cells appear red, while dead cells remain 6 https://mc06.manuscriptcentral.com/botany-pubs Page 7 of 27 Botany colourless. Germination recovery (%), which is the ability of the un-germinated seeds to germinate after being transferred from the saline solution to the distilled water was calculated as: Recovery percentage (%) = [ (a – b)/ (c – b)] × 100 where, a represents the number of germinated seeds within the saline solution and those that germinated after recovery in distilled water, b represents the number of germinated seeds in the saline solutions, and c represents the total number of seeds tested in the experiment (Khan and Ungar 1984). Statistical analysis The percentages of seeds that germinated are represented as mean ± SE. Data for germination and germination recovery percentage Draftwere arcsine transformed prior to statistical analysis to ensure homogeneity of variance. One-way analysis of variance (ANOVA) was used to determine significant differences between the treatments of temperature, light/dark, and saline solutions. Results Effects of temperatures and light/dark on germination After the 30-day incubation period, the percentages of germination were the highest at the lowest temperatures, however, they were lower at higher tempertures.