Zhaoyi Wang1, Sihui Tian1, 2, Jigao Wang1, Honggang Shuai1, Yaoyao Zhang1, Yuefeng Wang1, Baocheng Jin1 and Xuechun Zhao1,*
1 College of Animal Science, Guizhou University, Guiyang 550025, China;2 Institute of Botany, Chinese Academy of Sciences, Beijing 100000, China;
* Correspondence: xczhao@gzu.edu.cn; Tel.: +86-187-8600-8048
Abstract: Seed germination is critical for successful crop production, and the sensitivity to pH and salt stress depends on the plant’s tolerance mechanisms. In view of the characteristics of calcium-rich and acidic soils in the karst areas of Guizhou Province, China, the effects of pH stress and calcium stress on the seed germination characteristics of three herbages were studied with the goal of exploring and revealing the mechanism of adaptation of the three herbages to an acidic soil environment and providing a theoretical basis for the selection and cultivation of acid-tolerant herbages in southwest China. In this study, six concentration gradients of CaCl2, including 0, 25 mmol/L, 50 mmol/L, 100 mmol/L, 150 mmol/L, and 200 mmol/L, and seven pH gradients, including 4.55, 5.35, 6.61, 7.03, 8.0, and 9.18 were established, respectively. The germination rate, germination potential and germination index of the seedlings were measured for each seed germination and seedling growth stage of orchardgrass (Dactylis glomerata L.), perennial ryegrass (Lolium perenne L.), and alfalfa (Medicago sativa L.), respectively. The results showed that under the interaction of pH and CaCl2, the germination rate, germination potential and germination index of the three herbage seeds increased first and then decreased. When the concentration of salt stress began to change, the herbage seeds could adapt to salt stress at an appropriate pH condition. When only the pH value or CaCl2 concentration changes, the increase in pH and CaCl2 will inhibit the growth of shoots and roots. Weak acid can promote the growth of shoots and young roots, while alkaline conditions can inhibit their growth. The effect of a low concentration of CaCl2 was not apparent, while a high concentration of CaCl2 clearly inhibited the plants. The optimal pH and CaCl2 of the bud and root lengths changed after the interaction. In conclusion, there is a substantial difference between pH and calcium salt stress, and the interaction between pH and calcium salt concentration has a substantial influence on the salt and alkali tolerance of the three types of seeds.Keywords: seed germination; pH; calcium; germination rate; germination potential; germination index
Introduction
Seed germination and seedling growth are the most sensitive stages in plant life, representing the first contact with the environment, notably water and soil [1]. Rapid seed germination and stand establishment are critical factors that affect crop production under conditions of stress[2]. Salt stress, drought stress and alkaline stress can inhibit seed germination; yet there is a limited understanding of the potential interaction of these stresses, which often occur together in nature [3]. Guizhou Province lies in the heart of a karst region in southwest China, and its soil is acidic and rich in calcium salts [4,5]. Therefore, when seeds germinate in the soil, if the pH is too low, the phospholipid structure of the cell membrane will be destroyed; the permeability of the cell membrane will be changed, and the rate of degradation of stored substances in the seeds will be reduced. Simultaneously, the respiration intensity of seeds will be weakened, and the activity of hydrolases will be reduced, thus, inhibiting seed germination [6]. In contrast, higher pH values result in higher contents of OH-, which will interfere with the absorption of some key anions, affect the membrane potential energy, and inhibit the metabolism of seed storage compounds and related proteolytic enzyme activity to affect the germination of seeds [7]. As an important element in plant growth and development, calcium is involved in cell division and differentiation and the degradation and synthesis of membrane phospholipids among others. It is the primary regulator of plant metabolism and development. Ca2+ controls a series of important physiological processes and enzyme activities related to seed germination in cells [8]. Salinity has negative effects on the survival of plants. Seed germination and early seedling establishment can be strongly inhibited under salt stress, leading to significant reductions in plant density and poor growth, such as plant stunting, smaller and fewer leaves per plant, and a lower yield [9]. Salinity often generates osmotic stress and ion toxicity, which, in turn, cause nutritional deficiency and oxidative stress, further contributing to the restriction of plant growth, wilting or even death. Thus, salt stress is a limiting factor for improving crop yield, and the enhancement of salt tolerance is therefore, an important focus of current crop breeding programs [10]. Research shows that alkaline soils (a high pH from 8.5 to 11) endanger crop production more than soils that contain excess salt [11], To date, the effects of salt stress on plants have been widely reported [12,13], Understanding how plants respond to pH and calcium salt stress is essential for improving the tolerance of plants. Most studies on the effects of acid soils on forage seed germination have focused on growth, nodule formation, nitrogen fixation, and mineral nutrient uptake. Little attention has been paid to the sole effects of H+ toxicity or calcium on the germination and survival of seedlings of the Legumes and the Gramineae. The effects of solution pH and exogenous calcium on seed germination, seedling survival and the growth of herbage were reported in this study. In this context, this study aims to compare the response to pH and calcium salt stress of three forage plants commonly used in many countries as animal feed, including orchard grass (Dactylis glomerata), perennial ryegrass (Lolium perenne), and alfalfa (Medicago sativa). The physiology and biochemistry of seed germination and seedling growth were examined in an attempt to illuminate the effects of pH and calcium salt stress. In this context, under salt stress, the seed germination and seedling growth of M. sativa were inhibited by inappropriate concentrations, which led to a decrease in the germination rate [14]. Previous studies have clearly shown that the seed germination rate is always high in salt-free conditions but decreases as the concentration of soil salt increases. However, the interaction of salinity and alkalinity (pH) on seed germination remains unclear [15].
Materials and Methods
Seeds of D. glomerata, L. perenne, and M. sativawere purchased from Zhong Zhi Heng Seed Company (Guiyang, China) in October 2018 and stored dry in cloth bags at room temperature for further use. Seed germination experiments were conducted at the laboratory of Department of Grassland Science (26°44′ N and106°65′ E), Guizhou University (Guiyang, China).2.1 Experimental DesignA seed germination experiment was conducted in March 2019 using a petri dish filter paper hydroponic method to assess the combined effect of salt stress and pH on seed germination based on the characteristics of rich calcium and acid soil in the karst area in Guizhou Province. Six CaCl2 concentrations were established as follows: 0 (CCK), 25 mmol/L (C1), 50 mmol/L (C2), 100 mmol/L (C3), 150 mmol/L (C4), and 200 mmol/L (C5). There were six pH treatments, including 4.55 (pH1), 5.35 (pH2), 6.61 (pH3), 7.03 (pH4), 8.0 (pH5), and 9.18 (pH6), respectively. There were 36 treatments with three replicates (Table 1). Uniform and full-sized seeds of D. glomerata, L. perenne, and M. sativa were selected, sterilized with 0.5% KMnO4 for 20 min, and washed three times with distilled water. They were then sterilized with ethanol 70% for 30 s, rinsed three times with distilled water and arranged neatly in petri dishes covered with two sheets of filter paper. Different concentrations of CaCl2 and pH values were dripped on the seeds. Each per petri dish included 50 seeds, which were incubated at 25 °C in the dark. Each treatment was conducted in triplicate. The photoperiod was 12 h/day; the temperature was 25 ± 1 °C day and 14 ± 1 °C night, and the relative humidity was 80 ± 1%.Table 1. Design of the interaction between the concentration of CaCl2 and pH on the seeds of three forage plantsD. glomerata, L. perenne, and M. sativa