Research Paper the Effect of Salinity Stress and Temperature Levels

Research Paper

The effect of salinity stress and temperature levels on germination characteristics of four medicinal plants seed

Accepted 9th March, 2021

ABSTRACT

Cultivation of medicinal plants has been economically beneficial for pharmacy and medicine. These plants are stores of active and valuable secondary metabolites that can be converted into various drugs, some of which are life-saving. However, it is difficult to start a large-scale and commercial cultivation of these plants because most of the arable land is used to produce strategically essential crops. Other uncultivable lands are often affected by various abiotic stresses, one of the most important of which is salinity. Germination of plants is one of the important stages during their growth period that is often affected by environmental stresses, especially salinity. In this study, the seeds of medicinal plants such as Lavender (Lavandula angustifolia), hyssop (Hyssopus officinalis), black cumin (Nigella sativa L.) and Scrophularia (Scrophularia striata) were subjected to salinity stress at 20, 25 and 30°C in order to determine the germination characteristics of their seeds. The results of this experiment generally showed that with increasing salinity stress at different temperatures, all germination characteristics including germination percentage, germination rate, shoot length, root length, shoot dry weight, root dry weight and seed vigor index decreased. It seemed that in order to Yousef Hakimi grow plants commercially, low salinity soil and water are needed to get the best yield. Department of Horticulture, Faculty of Agriculture and Natural Resources, University Key words: Medicinal plants, salt stress, seed treatment, germination, lavender of Tehran, Karaj, Iran. (Lavandula angustifolia), hyssop (Hyssopus officinalis), black cumin (Nigella sativa Email: [email protected] L.), Scrophularia (Scrophularia striata).

INTRODUCTION

Seed is the main factor for reproduction and preservation In addition to serving as a protective barrier during of plant hereditary reserves, which plays an important role embryogenesis, seeds also provide nourishment during in the distribution and establishment of plants in different both embryogenesis and early seedling development (Taiz areas, preservation and survival of plant generation in et al., 2015). adverse and long-term conditions (Bewley et al., 2013a). Approximately 20% of the world's cultivated areas and Seed germination is a physiological process in which the about half of the irrigated land are affected by salinity, and embryo emerges from the end of the enclosing coverings, its amount increases daily (Flowers, 2004). Salinity is the which can include the endosperm, perisperm, testa, and presence of excessive soluble salts and minerals in the soil pericarp. Germination begins with the absorption of water and water environment in which plants are not able to by the seed and ends with the exit of the embryonic axis, absorb enough water, which disrupts the metabolism and including the root and stem, through the surrounding physiological activities of plants, lack of absorption of structures (Bewley et al., 2013b). The ability of seeds to nutrients and disorders of photosystems 1 and 2 (PSI and delay germination through the dormant mechanism is one PSII), reduce leaf development as well as plant growth, of the most important ways to maintain survival in plants. development and yield (Munns and Tester, 2008). Usually Academia Journal of Medicinal Plants; Hakimi. 071

the highest sensitivity to salinity is observed in the life cycle Cl− during salt stress have resulted in decreased levels of N, of plants during germination and at the beginning of P, K+ , Ca2+ and Mg2+ in fennel, Trachyspermum ammi, seedling growth (Kermode, 1990). The study of the effects peppermint, lemon verbena, Matricaria recutita, Achillea of salinity on germination rate and germination percentage fragratissima (Abd EL-Azim and Ahmed, 2009; Abd El- as well as root and shoot growth in many plants has shown Wahab, 2006; Oueslati et al., 2010). Ali et al. (2013) showed that salinity stress at the germination stage is a reliable test that the N, P and K+ percentages were influenced by in assessing stress tolerance in many species as salinity different salt concentrations in the leaves of Simmondsia reduces the percentage and speed of germination and also chinensis (jojoba). Salt concentration over 17.2 mM reduces the root and shoot length (Ghoulam and Fares, drastically lowered the three elements. Similar decrease 2001). was reported for the Ca2+ content in the salt stressed During the early stages of salinity stress, due to osmotic leaves. In the literature, it has been shown that when stress which results due to high accumulation of salt in soil sowing seeds in soils with high salt content, germination in and plants, the water uptake capacity of root systems is Ocimum basilicum, Eruca sativa, Petroselinum hortense, reduced and water loss from leaves is accelerated, hence chamomile, sweet marjoram and Thymus maroccanus is salinity stress is also considered as hyperosmotic stress reduced and delayed (Ali et al., 2007; Belaqziz et al., 2009; (Munns, 2005). Plants increase ROS production in response Miceli et al., 2003; Ramin, 2006). The aim of this study was to salinity stress, such as singlet oxygen, superoxide, to investigate the effect of salinity stress on germination hydroxyl radicals, and hydrogen peroxide (Ahmad, 2010; characteristics of seeds of Lavender (Lavandula Apel and Hirt, 2004; Mahajan and Tuteja, 2005). The angustifolia), hyssop (Hyssopus officinalis), black cumin formation of ROS due to salinity stress can lead to oxidative (Nigella sativa L.) and Scrophularia (Scrophularia striata). damage to various plant cell components such as lipids, proteins and DNA and disrupt the vital functions of plant cells (Gupta and Huang, 2014). Materials and methods In the last two decades, a great deal of research has been done to understand the molecular and physiological This study was carried out at the Department of mechanisms of salinity tolerance in Arabidoboxis (Zhang Horticulture Science, College of Agriculture and Natural and Shi, 2013). Salinity tolerance includes a set of Resources, University of Tehran, Karaj 31587, Iran. Seeds of responses at the molecular, cellular, metabolic, medicinal plants from IPK Gatersleben Company were used physiological, and whole plant levels. Extensive research as seed material. The temperature regime used consisted of has been done through cellular, metabolic and physiological three levels (20, 25 and 30 °C). At each temperature, in analysis; the results of these studies have shown that order to create salinity stress, five levels of salinity among the various reactions related to salinity stress, ion potential including 0, 50, 100, 150 and 200 mM bar were absorption control, osmotic regulation, hormone applied, which were prepared using sodium chloride salt metabolism, antioxidant metabolism and stress signaling with the required amounts. Also, distilled water was used mechanisms or strategies play an important role in plant to create zero stress level (control) in each experiment. adaptation to salinity stress (Gupta and Huang, 2014). Salinity directly inhibits plant growth by disrupting the nutrient uptake equilibrium maintained by the plant Seed treatment system. Nutrient availability, partitioning and transport are mostly affected. This is due to the competition of Na+ and 50 seeds were disinfected with 5% sodium hypochlorite − + 2+ − Cl ions with the nutrient ions like K , Ca and NO3 . Such solution for 30 sec and after rinsing with distilled water, ionic imbalances caused due to the abundance of Na+ and they were transferred to filter paper in petri dishes. Then, Cl− ions directly acts upon the biophysical and/or metabolic for salinity treatment, 10 ml of sodium chloride solution components of the plant system (Banerjee and was added to each petri dish and after applying the Roychoudhury, 2016). treatments at the time of sowing, the petri dishes were Medicinal and aromatic plants are valuable products; the placed in a germinator with a relative humidity of 80% and natural products of these plants are small volume but very after applying the treatments at the time of sowing the valuable and have many applications in various industries seeds, the petri dishes were placed in a germinator with a such as food, beverages, food supplements, perfumery, relative humidity of 80% and temperatures of 20, 25 and cosmetics and medicine (Novak and Blüthner, 2020). 30°C, 16 h of light and 8 h of darkness. Salinity stress has been reported as a major inhibitor of plant growth in case of medicinal plants like Majorana hortensis, peppermint, pennyroyal, apple mint, Aloe vera, Germination tests Matricaria recutita, Thymus maroccanus, geranium, Thymus vulgaris, sweet fennel, sage and Mentha pulegium (Aziz et The criterion for seed germination was 2 mm root al., 2008; Said-Al Ahl and Omer, 2011). Increases in Na+ and emergence. Petri dishes were inspected at a specific time Academia Journal of Medicinal Plants; Hakimi. 072

and the number of germinated seeds was counted to was obtained in the treatment of non-stress and determine the germination rate daily up to 14 days after temperature of 25°C and the lowest amount except for planting and to add water or salt solution as necessary to treatments that did not have any germination was 7%, 7.41 maintain an adequate moisture level. On the fourteenth and 4.66 respectively in the salinity level 150 mM and 20°C. day, 20 seedlings were selected from each petri dish and No germination was observed in salinity treatment of 150 traits such as germination percentage and speed, root and mM and temperature of 30°C and salinity treatment of 200 shoot length and seed vigor were measured (Ellis and mM as shown in Table 2. The highest Plumule Length (200 Roberts, 1981). Additional samples of each treatment were mm) and Radical Length (310 mm) was obtained in the used to visually estimate abnormal seedling development, treatment of non-stress and Temperature of 25°C and the according to ISTA (ISTA, 1999) rules, at 10 days after lowest amount except for treatments that did not have any initiating germination. To calculate the germination germination was 58 and 75 mm respectively in the salinity percentage, Formula GP=100(Ni/N) was used. Where GP is level 150 mM and 20°C. the germination percentage, Ni is the number of germinated seeds until day i and N is the total number of seeds (Hartmann and Kester, 2014). Germination rate was also Hyssop (Hyssopus officinalis) calculated through Equation RS= (ni·t(x-i))/ni. In this formula, RS is the germination rate (number of seeds per The highest Germination percentage (83.00%) and Seed day), n is the number of seeds germinated on day i, and x is i Vigor Index (19.53) was obtained in the treatment of 50 the total number of days of the test (Bewley et al., 2013b). mM NaCl and Temperature of 20°C and the lowest amount In addition seed vigor using Relationship V= (GP/100) × except for treatments that did not have any germination (퐿푟 + 퐿푠/2) was calculated. In this regard, V is the seed was 51% and 2.08 respectively in the salinity level 150 mM vigor index, Lr is the Radical Length, Ls is the Plumule and 30°C. No germination was observed in salinity Length and GP is the Germination percentage. treatment of 200 mM NaCl as shown in Table 4. The highest

Plumule Length (30.40 mm) and Radical Length (16.63 Experimental design mm) was obtained in the treatment of 50 mM NaCl and Temperature of 20°C and the lowest amount except for The experimental design was two factors factorial (5 × 3) treatments that did not have any germination was 4.80 arranged in a completely randomized design; with three (mm) in salinity level 150 mM and 30°C and 75 (mm) in replications and 50 seeds per replicate. The first factor was salinity level 150 mM and 20°C respectively. seed treatments (salinity potential including 0, 50, 100, 150 and 200 mM); the second was temperature levels (20, 25 and 30°C). Data for germination and abnormal germination Black cumin (Nigella sativa L.) percentage were subjected to arcsine transformation before analysis of variance was made using SPSS Statistics The highest Germination percentage (64.00%) and Seed 26 program (IBM). The differences between the means Vigor Index (23.67) was obtained in the treatment of 50 were compared using Duncan values (P < 0.05). mM NaCl and Temperature of 25°C and the lowest amount

was 2% and 0.05 in the salinity level 200 mM and 30°C as RESULTS shown in Table 6. The highest Plumule Length (40 mm) and Radical Length (41 mm) was obtained in the treatment of Reduction of water uptake by seeds due to salinity stress non-stress and Temperature of 25°C and the lowest was 3 reduces physiological and metabolic processes and and 2 mm respectively in the salinity level 200 mM and therefore the abundance of materials available for plant 30°C. survival is problematic and reduces germination (Ashraf and Waheed, 1990). The results of analysis of variance showed (Table 1, 3, 5 and 7) that salinity stress had a Scrophularia (Scrophularia striata) significant effect on the evaluated traits and with increasing Salinity stress, Germination percentage, Germination rate, The highest Germination percentage (84.00%), Plumule Length, Radical Length, Plumule Dry weight, Germination rate (47.62) and Seed Vigor Index (40.95) was Radical Dry weight and Seed Vigor Index decreased obtained in the treatment of non-stress and temperature of significantly. 25°C and the lowest amount was 4%, 20.83 and 0.42 respectively in the salinity level 200 mM and 30°C (Table 2). The highest Plumule Length (58.5 mm) and Radical Lavender (Lavandula angustifolia) Length (39 mm) was obtained in the treatment of non- stress and Temperature of 25°C and the lowest amount was The highest Germination percentage (75.00%), 9 and 6 mm respectively in the salinity level 200 mM and Germination rate (15.38) and Seed Vigor Index(191.25) 20°C. Academia Journal of Medicinal Plants; Hakimi. 073

Table 1: Results of analysis of variance of studied traits under salinity stress and Temperature treatment in Lavender.

Plumule Radical Seed Germination Germination Plumule Radical SOV df Dry Dry Vigor percent rate Length Length weight weight Index Salinity 4 7739.30** 328.34** 59908.70** 146407.70** 191.19** 1.93** 49274** Temperature 2 36.60** 21.70** 1503.2** 1854.2** 4.21** 0.11* 329.92** Salinity×Temperature 8 14.60** 10.77** 795.70** 1183.70** 0.462* 0.03 70.76** E 30 0.73 0.73 0.73 0.73 0.18 0.03 1.11-28 CV (%) 95.74 65.81 72.52 76.12 85.76 75.39 115.9

**significant differences at 1%, * significant differences at 5%.

Table 2: Comparison of the mean interaction of salinity levels × temperature on the studied traits in Lavender.

Plumule Radical Seed Temperature Germination Germination Plumule Radical Salinity (mM) Dry Dry Vigor (°C) percent rate Length Length weight weight Index

20 71.00b 13.33b 183.00c 292.00c 10.50b 0.95abc 168.63c 0 25 75.00a 15.38a 200.00a 310.00a 11.25a 0.97abc 191.25a 30 72.00b 13.89ab 190.00b 300.00b 10.00b 0.85bcd 176.40b 20 36.00d 12.35bcd 138.00f 243.00f 8.75c 1.15ab 68.58f 50 25 40.00c 13.89ab 150.00d 260.00d 9.25c 1.20a 82.00d 30 39.00c 12.66bc 140.00e 248.00e 8.00d 1.07ab 75.66e 20 21.00g 11.36cd 107.00i 160.00i 4.25e 0.73cde 28.04i 100 25 25.00e 12.50bcd 120.00g 170.00g 4.50e 0.65cdef 36.25g 30 23.00f 10.99d 110.00h 161.00h 3.75e 0.60def 31.17h 20 7.00h 7.41e 58.00k 75.00k 1.50f 0.38ef 4.66k 150 25 8.00h 7.75e 70.00j 80.00j 2.00f 0.48f 6.00j 30 ------20 ------200 25 ------30 ------

In each column, the means with at least one common letter using Duncan's test at the 5% probability level are not significantly different. Academia Journal of Medicinal Plants; Hakimi. 074

Table 3: Results of analysis of variance of studied traits under salinity stress and Temperature treatment in hyssop.

Plumule Radical Seed Germination Germination Plumule Radical SOV df Dry Dry Vigor percent rate Length Length weight weight Index

Salinity 4 9613** 209.15** 1049.61** 256.30** 7.02** 0.07** 397.5** Temperature 2 274.40** 2.05 8.83** 22.24** 0.13* 0.01* 20.05** Salinity×Temperature 8 70.15** 0.86 8.30** 4.48** 0.01 0 2.87** E 30 0.80 0.80 0.80 0.81 0.03 0 0.33 CV (%) 53.24 56.69 75.55 74.34 78.85 61.54 82.88

**significant differences at 1%, * significant differences at 5%.

Table 4: Comparison of the mean interaction of salinity levels × temperature on the studied traits in hyssop.

Plumule Radical Seed Temperature Germination Germination Plumule Radical Salinity (mM) Dry Dry Vigor (°C) percent rate Length Length weight weight Index 20 78.00b 11.36abc 19.10d 11.20bc 2.10ab 0.19cd 11.82c 0 25 79.00b 13.00a 16.00e 8.40e 2.20a 0.20bcd 9.64d 30 67.00e 12.66ab 18.80d 8.70de 2.00abc 0.17de 9.21d 20 83.00a 10.31c 30.40a 16.63a 1.80cd 0.23ab 19.53a 50 25 82.00a 12.19ab 25.70c 12.30b 1.90bc 0.24a 15.59b 30 79.00b 10.99bc 27.60b 12.40b 1.60d 0.22abc 15.79b 20 75.00c 7.94d 14.70e 10.00cd 0.90e 0.15ef 9.26d 100 25 63.00f 8.50d 14.9e 6.70f 1.00e 0.13fg 6.81e 30 66.00e 8.13d 11.40f 5.80f 0.80e 0.12fgh 5.68f 20 69.00d 7.90d 5.60h 3.30g 0.50f 0.09hi 3.06g 150 25 53.00g 7.50d 7.30g 3.50g 0.50f 0.10ghi 2.87gh 30 51.00h 7.30d 4.80h 3.40g 0.30f 0.08i 2.08h 20 ------200 25 ------30 ------

In each column, the means with at least one common letter using Duncan's test at the 5% probability level are not significantly different. Academia Journal of Medicinal Plants; Hakimi. 075

Table 5: Results of analysis of variance of studied traits under salinity stress and Temperature treatment in black cumin.

Plumule Radical Seed Germination Germination Plumule Radical SOV df Dry Dry Vigor percent rate Length Length weight weight Index

785.5 Salinity 4 4637.86** 276** 1851.7** 2301.5** 25.57** 0.22** 7** 37.13* Temperature 2 162.16** 77.6** 60.80** 39.20** 0.39* 0.01 * Salinity×Temperature 8 8.66** 5.1** 2.05 0.95 0.03 0.001 5.59** E 30 1.09 1 1 1 0.09 0.002 0.08 CV 58.82 27.96 62.69 70.44 73.81 356.4 87.41

**significant differences at 1%, * significant differences at 5%.

Table 6: Comparison of the mean interaction of salinity levels × temperature on the studied traits in black cumin.

Plumule Radical Seed Germination Germination Plumule Radical Salinity (mM) Temperature (°C) Dry Dry Vigor percent rate Length Length weight weight Index

20 49.00d 24.00c 38.00b 39.00b 4.20ab 0.43ab 18.87c 0 25 52.00c 32.00a 40.00a 41.00a 4.50a 0.39bc 21.06b 30 44.67f 26.00b 36.00c 38.00b 4.00abc 0.35cd 16.52cd 20 61.00b 20.00ef 33.00d 35.00c 3.50cd 0.46ab 20.73b 50 25 64.00a 26.00b 36.00c 38.00b 3.70bcd 0.48a 23.67a 30 53.00c 22.00d 30.00e 33.00d 3.20d 0.43ab 16.69cd 20 45.00f 18.00gh 19.00g 16.00f 1.70e 0.29de 7.88f 100 25 47.00e 21.00de 21.00f 18.00e 1.80e 0.26ef 9.17e 30 41.00g 19.00fg 16.00h 15.00f 1.50e 0.24efg 6.36g 20 21.00i 14.00ij 11.00i 9.00h 0.80f 0.17gh 2.09i 150 25 24.00h 17.00h 12.00i 11.00g 0.80f 0.19fgh 2.77h 30 18.00j 15.00i 9.00j 8.00h 0.60f 0.15hi 1.53j 20 4.00k 13.00j 5.00k 3.00ij 0.40f 0.08ij 0.17k 200 25 4.00k 15.00i 5.00k 4.00i 0.45f 0.08ij 0.18k 30 2.00l 13.00j 3.00l 2.00j 0.35f 0.07j 0.05k

In each column, the means with at least one common letter using Duncan's test at the 5% probability level are not significantly different. Academia Journal of Medicinal Plants; Hakimi. 076

Table 7: Results of analysis of variance of studied traits under salinity stress and Temperature treatment in Scrophularia.

Plumule Radical Seed Germination Germination Plumule Radical SOV df Dry Dry Vigor percent rate Length Length weight weight Index

Salinity 4 51.73.7** 786.62** 2610.33** 1152.09** 25.57** 0.21 1449.84** Temperature 2 1897.80** 157.62** 6.17 6.27** 0.39 0.01 192.03** Salinity×Temperature 8 76.55** 2.87* 26.99** 12.21** 0.03 0.001 35.97** E 30 1.00 1.00 3.89 1.00 1.00 0.25 0.48 CV 58.87 27.84 51.62 51.87 56.13 57.14 93.75

**significant differences at 1%, * significant differences at 5%=.

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