Effect of Saline-Alkaline Stress on Seed Germination and Seedling Growth of Triticale

Effect of Saline-Alkaline Stress on Seed Germination and Seedling Growth of Triticale | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of Saline-Alkaline Stress on Seed Germination and Seedling Growth of Triticale Yu Xiu, Rao Fu, Shuhui Ma, Qitong Huang, Lifeng Hua, Lei Nie, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7025898/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract To investigate the adaptation ability of triticale to saline-alkaline stress, SN-1 triticale was used in this study to determine the roles of different saline-alkaline ratio stress treatments during seed germination to seedling stage. In this study, we used two neutral salts (NaCl and Na 2 SO 4 ) and two alkaline salts (Na 2 CO 3 and NaHCO 3 ), mixed in 25 different ratios, We treated SN-1 under these 25 different saline-alkaline conditions and tested seed germination and seedling stage physiological indexes including germination percentage, germination energy, germination index, seedling stem length, seedling root length, the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), soluble proteins, etc. The results showed that during the period of merging, when pH and salt concentration were low, germination percentage and potential were increased. The treatment groups showed similar changing trends. The length of the germ and radicle decreased when salt-alkali concentration and especially the pH were increased, and the radicle was more sensitive than the germ. The activity of protective enzymes SOD, POD and CAT increased when salt-alkali content was low. Soluble protein content initially increased and then decreased when salt-alkali content increased. In photosynthesis system, Chlorophyll a increased while Chlorophyll b decreased. The root was very sensitive to salt-alkaline stress, all indexes showed the same trend of initial increase and subsequent decrease. The root superficial area, length, root tip number showed the same trend and decreased rapidly. All the physiological indexes indicated that the interactive effect of pH and salt concentration must be considered before sowing triticale in salt-alkaline land. black wheat mixed saline-alkaline stress seed germination seedling growth activity of protective enzymes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background The triticale with blue, purple-black, black, etc., grain color is a new variety of wheat, which has the advantages of turning green quickly, drought resistance, etc. The protein content is high and can reach 17.1–20.5% (Zhang long Yu, etc.). The content of essential amino acids such as isoleucine, leucine, etc., is 33.3–75.0% higher than the white wheat(Rong Li,etc.1995). Moreover, the contents of trace elements such as selenium, iodine, iron, etc., are higher than the white wheat(Yeo A R.1999; Ashraf M,2005; Ashraf M.2009). The anthocyanin content is also high(Xiao lan Li,etc.), and has active ingredients such as flavone, polysaccharides, etc., which have various pharmacological effects, such as free radical elimination, anti-aging, blood glucose reduction, etc(ZILIC S,etc.2011;BABBAR N,etc.2011;WAGNER J R,etc.1992). Hence, triticale is a health product with high nutritive value and wide prospective market applications(Zhang long Yu, etc.2016;Zhang long Yu,etc.2020;Yeo A R.1999). The saline-alkaline stress is one of the most important factors that affects the agriculture and land productivity. Moreover, 25% of global land area is affected by salinization(Ashraf M.2005;Ashraf M.2009; Asish Kumar Parida,etc.2005).The saline-alkaline area has increased at the speed of (1-1.5)×106 hm2 because of unreasonable irrigation, excessive use of chemical fertilizers, destruction of forests and vegetation, and global warming due to greenhouse effects, which have severely threatened the ecological environment and affected the sustainable development of agriculture(Darwish T, etc.2005; Lä uchli A,etc.2008). The northeast and northwest of China have saline-alkali land, comprising of alkaline salts (NaHCO 3 and Na 2 CO 3 ), and neutral salts (NaCl and Na 2 SO 4 ). The salinization and alkalization occurred simultaneously(Fen Fen Xu,etc.2012;Xiao Lei Zhang,etc.2013) producing salt and alkaline stress, and the alkaline salts have high pH, which could cause more serious harm to crops (Jian Xin Liu,etc.2016;Ya Ci Lv2021;Ya qi Chen,etc.2021). Therefore, salt stress and alkaline stress are research hotspots, but previous studies have always focused on either salt or alkaline stress, or comparison between them, and the combined saline-alkaline stress has been rarely studied. The influence of saline-alkaline stress on triticale germination and seedling growth stages has been rarely studied at home and abroad. Hence, we investigated the triticale SN-1 response to saline-alkaline stress during the germination and seedling growth stages to uncover the salt tolerance mechanism by determining the physiological indexes and active substance content changes, which provided a theoretical basis to uncover the molecular mechanism on the microcosmic level and the possibility of cultivating triticale on saline-alkaline land, leading to wide market application prospects for triticale. Materials and Methods Experimental Design 1. Mixed saline-alkaline condition simulation We chose two neutral salts (NaCl and Na 2 SO 4 ) and two alkaline salts (Na 2 CO 3 and NaHCO 3 ), mixed in different ratios(A: NaCl:Na 2 SO 4 1:1, B: NaCl:NaHCO 3 1:1, C:NaCl:Na 2 SO 4 :NaHCO 3 :NaCO 3 1:1:1:1, D:Na 2 SO 4 :NaCO 3 1:1, E:NaHCO 3 :NaCO 3 1:1), that were 25 different saline-alkaline conditions. We treated triticale SN-1 under these 25 conditions and tested seed germination and seedling stage physiological indexes. 2. River sand experiment The experiment was conducted in the greenhouse provided by Shandong Silkworm Institute, Shandong, China, in 2021. The plastic pots used were 20cm in diameter and 30cm in height. They were filled with 300g river sand, and placed in the hydroponic box with Hoagland, to immerse the river sand with Hoagland. The nutrient solutionwas replaced after five days. The mode was irrigation and the experimental period was four weeks. We picked the plants with uniform growth for next period that was saline-alkaline stress.The hydroponic box was filled with 1500ml of each saline-alkaline solution, irrigated for one week, and then the triticale seedlings were used for testing. Experimental Methods Stress treatment For sowing triticale in late September 2021, first all seeds were disinfected with hypochlorite for 15 minutes, then rinsed with water until clean, and dried. The dried seeds were planted at a depth of 1- 2cm and a density of 15 seeds in the plastic pots with 300g river sand and then placed in the hydroponic box with all Hoagland.The selected 25 plastic pots were treated daily with about 1000 ml of 1∕2 Hoagland solution containing the above-described ratios of mixed saline-alkaline solutions to simulate natural saline-alkaline stress,while the same volume of nutrient solution without additional saline-alkaline solution was applied to the control group. All the pots were irrigated with nutrient solution and then treated by saline-alkaline stress for one week. Each saline-alkaline solution was added at 5:00–6:00pm daily for one week. Recording of seed germination and survival We picked some triticale to test the germination, and made the above-mentioned 25 different ratios of saline-alkaline solutions and used them to simulate saline-alkaline conditions, the control group was treated with water, and every concentration had three repeats. The seeds were disinfected with hypochlorite for 15 minutes, rinsed with water until clean, and then placed on the petri dish (Φ = 7 cm). The petri dish had two layers of filter paper, with 30 seeds on the filter paper. The petri dishes were placed in a light incubator. The germination standard was when the length of the radicle was twice than that of the seed, and the length of the germ was equal to the seed (Hong wei Yang,etc.2017). We started counting the germinated seeds from the second day after sowing, and when the germination number remained the same for three days, it was regarded as the end of germination, The germination was continued for 10 days, and then the length of the radicle and germ were tested. Seed germination index : Germination percentage = number of germinated seeds on the fifth day∕number of tested seeds× 100% (the germination number of every group remained the same five days later),Germination potential = number of germinated seeds on the second day∕number of tested seeds× 100% ( the daily germination number of the control group peaked on the second day) Measurement of seedling growth and other physiological parameters On the 35th day after sowing, the height and wet weight of three randomly selected seedlings were measured, and each measurement was repeated 3–5 times. Mechanisms of resistance in the seedling Under the saline-alkaline stress condition, the growth and development of plants are inhibited, and the plasma membrane is most seriously affected with the increase in concentration, the extravasation of electrolyte is increased, the free radical metabolism balance in the cell is destroyed(De zong Sui,etc.2007; Jing Tao.; Guo Qiang Wu,etc.2017), and the membrane lipid peroxides destroy the membrane system. malondialdehyde (MDA) was one of the production of the membrane lipids peroxide product, so the concentration of MDA increased that was the result of destruction of the cell membrane lipids and also the reason of destruction, the concentration of MDA reflected the cell membrane destruction degree and intensity of the reaction to the adversity condition (Jiu Ju Duan,etc.2006;Jun Guo Zhou,etc.2007;Chun mei Han,etc.2009). Plants produce large amounts of reactive oxygen species (ROS) due to saline-alkaline stress, because of resisting the destruction of the ROS, the plants established an effective ROS eliminating system during evolution, which could maintain the dynamic balance of ROS. The system comprises of an enzymatic antioxidant system and a non-enzymatic antioxidant system. The enzymatic antioxidant system comprises of SOD, CAT, POD, etc., while the non-enzymatic antioxidant system comprises of ascorbic acid (As A) and glutathione (GSH). The activity and concentration of antioxidant enzymes and the concentration of antioxidant material could reflect the elimination ability of ROS or intensity of resistance to adversity(Yan ru Guo,etc.2006). Measurement of root length and other structural parameters The root system has important influence on plants by absorbing and transporting water and nutrients in the soil and synthesizing and storing nutrition (Parida A K,etc.2005). The root system distribution affects the absorbance of nutrients and water from the soil, as well as the growth and developing ecology function setted up aboveground. Photosynthesis parameters Photosynthesis is one of the most important physiological processes for plant growth and development(Munns R,etc.2008). Photosynthesis pigments facilitate the plant light reaction(Petrov V V,etc.2014),and chlorophyll is the main photosynthesis pigment (Ru La Sa,etc.2014).Saline-alkaline stress can destroy the photosynthesis system in a dose-response manner. Fresh leaves were collected, washed with distilled water, dried, shredded and mixed well.Thereafter, 0.2g of the leaves were weighed, added to 25ml calibration tubes, and treated with 10ml of 95% ethanol. The tubes were capped and placed in 60–80 ℃ water for extracting chlorophyll, until the green color of the leaves had faded. The tubes were cooled and 95% ethanol was added to scale, and the supernatant was tested for chlorophyll content. Statistical analysis All the presented data were the mean values of three replicates and expressed as mean ± SD. The data were analyzed using the SPSS 21 .0 software. The level for significant difference was p<0 .05 . Results Seed germination process and final germination percentage of triticale Under saline-alkaline stress, seed germination is the key index for successful breeding of resistant varieties(Xu Zhao, etc).Generally, with increasing salt concentration, the water potential around the seeds decreases and the difference in water potential between the interior and exterior of the seed also decreases, limiting water absorption by the seeds.Insufficient water absorption impairs the utilization of the material stored in the endosperm and inhibits respiration, limiting seed germination, and results in a decreased final germination percentage and delay in seed germination. This study showed that saline-alkaline stress significantly affected the mean time of germination, germination percentage, germination potential and germination index .With the increase in salt-alkali concentration, the germination percentage, germination potential and germination index of the same treatment group decreased. When the alkaline stress increased, the reduction of germination parameters was maximum. When the concentration was 150mmol·L - 1 , the germination percentage, germination potential and germination index of the treatment group C were 60%, 46% and 20% ,respectively.When the concentration reached 200mmol·L - 1 , the germination percentage, germination potential and germination index declined to 20%, 57% and 36% ,respectively. The germination percentage of the treatment group C declined to 9% at 150mmol·L - 1 compared to 100mmol·L - 1 , the treatment group D declined to 49%, the germination potential of the treatment group C declined to 18% at 150mmol·L - 1 compared to 100mmol·L - 1 and the treatment group D declined to 63%. It was observed that alkalinity seriously affected the germination percentage and germination potential, and seriously limited merging compared to the neutral salt. When the concentration reached 300 mmol·L - 1 , every treatment group except group B had no merging, and the treatment groups D and E showed similar changes. With the increase in alkali concentration, the height of the gemmule and radicle of the same treatment group decreased. When the concentration reached 100mol·L - 1 , the height of the gemmule of the treatment group C was 63.14 mm, and when the concentration reached 150mmol· L - 1 , declined by 68%, the height of gemmule of the treatment group C declined by 9% of 100 mmol·L - 1 . Compared to 50mmol·L - 1 , the treatment group D declined by 66% ; when the concentration reached 50mmol· L - 1 , the height of radicle of the treatment group C was 25.91mm. When the concentration reached 100mmol·L - 1 , the height of the radicle of the treatment group C declined by 63% of 100 mmol·L - 1 . Compared to 50mmol·L - 1 , the treatment group D declined by 83%. Hence, alkalinity seriously affected thegemmule and radicle compared to neutral salt. When the concentration reached 300mmol·L - 1 , the gemmule did not merge except in the treatment groups A and B, and only the radicle of the treatment group A merged. The height of the radicle in the treatment groups D and E showed the same change. Seedling growth and other physiological parameters A previous study showed that the interactive effect of pH and salt significantly inhibited the height and dry weight of the entire plant, and that of roots, stems, and leaves, and the differences between any two groups were significant (Kai li Liu,etc.2005). Our study showed that height and wet weight of the plants in the treatment groups C, D and E rapidly decreased when the saline-alkaline concentration increased.This phenomenon was obvious in the treatment group E, and the wet weight and height of the plant were significantly affected by pH but not salt concentration .Hence, it can be concluded that when subjected to saline-alkaline stress, alkalinity was the major factor, and the wet weight and height of the plant decreased obviously with the increase in pH, which was significant in the treatment group E. Protective enzyme activity Saline-alkaline stress leads to extensive ROS production in plants that increases membrane permeability and lipid peroxidation, as well as leakage of electrolytes, which are harmful to the cytoplasmic membrane of plants(Dian Wang,etc.2012; Schreiber U,etc.1986).Plants secrete organic and inorganic substances to maintain their metabolic balance and synthesize protective enzymes to scavenge ROS, which enables the plant to tolerate saline-alkaline stress or reduce the damage caused by ROS(Liang Q,etc.2007). In this study, when triticale were cultured in 50mmol·L - 1 salt-alkali concentration, the activities of SOD, POD and CAT increased compared to the control group. The antioxidant enzyme activity decreased significantly under high pH and salt concentration in every treatment group, and this trend was more remarkable under high pH, suggesting that pH was the main influencing factor compared to salt concentration, which implied that the plants had limited tolerance when challenged with saline-alkaline stress, and if the concentration exceeded the plant ’s endurance limit, the long-term effects of ROS could inhibit the growth and survival of plants. Soluble protein content The concentration of soluble proteins showed a different trend compared to antioxidant enzyme activity.With increase in pH, the concentration of proteins increased and subsequently decreased, implying that under salt stress, more enzymes could be produced by plants to reduce the harm to plants. However, accumulation of ROS could damage the plants, which affected antioxidant enzyme production after the salt concentration was increased and then decreased. Root structure parameters When subjected to saline-alkaline stress, the morphological and physiological characteristics of the plants changed. Root formation of plants is based on hereditary characteristics, and restricted by soil ecological environment. In adverse situations, the root could change its form and distribution to adapt to harmful environment. A large study indicated that salt stress could limit root elongation and side root development, which can reduce root length, superficial area, and volume. Our study showed that when the salt concentration increased, the root superficial area, length, volume, and root tip number decreased, but not the average root diameter. With the increase in salinity, all parameters showed the same trend of initial increase followed by decrease, indicating that plants had certain tolerance which reduced the harm caused by salt. Among all the treatment groups, the treatment group E with the highest pH had the lowest values of all indexes. Photosynthesis parameters To protect itself from lack of moisture absorption and light damage caused by the saline-alkaline stress, the plant reduces light absorption and improves its energy conversion efficiency to ensure carbon assimilation, keeping the leaf temperature relatively low.Chlorophyll a is the core pigment for photosynthesis that transforms solar energy into photochemical energy. Therefore, its content reflects the level of light energy conversion efficiency. Chlorophyll b is a light-harvesting pigment that is responsible for light absorption and transmission. Thus, regulation of the contents of chlorophyll a and b, as well as the chlorophyll a∕b ratio, is an important regulatory mechanism for resistance in the plant. In this study, chlorophyll a showed an increasing trend with the increase in saline-alkaline content, whereas chlorophyll b content decreased, which resulted in a higher chlorophyll a∕b ratio than that in the control group. These results indicated that saline-alkaline stress increased the core photosynthetic pigment but reduced the light-harvesting pigment,thus ensuring normal photosynthesis in triticale. Conclusion In order to examine the influence of salt-alkaline stress on triticale during the period of merging and seedling, we selected triticale SN-1 as the study subject for investigating the effect of salt-alkaline stress on the root growth during germination in river sand so that that the root can be easily washed. We started sowing in late September 2021 in a greenhouse in Shandong Silkworm Institute. The entire process was continued for one month and one week. In the beginning,irrigating nutrient solution was used for one month. Thereafter, different ratios of salt-alkaline solutions were added for one week(Mao Q Z,2008; Lynch J P.1995; Yi L P,etc.2006). At the trefoil stage, we collected the triticale blade, and detected the physiological and biochemical indexes to investigate the effects on SN-1. The results showed that during the period of merging, when pH and salt concentration were low, the germination percentage and potential were increased, the interactive effect of pH and salt concentration was more significant, which resulted in rapid decline in germination percentage.Therefore, low salt concentration could promote plant merging, the plant had some resistance to stress, but this resistance disappeared when the concentration was increased(Tong H,etc.2012; Saveyn A,etc.2010). The treatment groups D and E showed similar changing trends, indicating that high pH was the main influencer.The lengths of the germ and radicle decreased with the increase in salt-alkali concentration. When the pH was high, the radicle was more sensitive than the germ(Chu Wu,etc.2004). During the period of seedling, the height and wet weight of the plant showed similar trend, but unremarkable compared to the germ and radicle(Guo Cong Xu,etc.2016; Khan, M.A.etc.2003; Ahan, J.etc.2009), which indicated that seedling resistance was stronger than seeds in the merging period; activity of the protective enzymes SOD, POD and CAT increased when content was low(Zhang, X. Q.etc.2013). When pH and salt concentration were increased, the activity decreased, suggesting that at the beginning of stress, plants had some tolerance, which gradually decreased and eventually disappeared. The soluble protein content increased and subsequently decreased with the increase in salt-alkali content, indicating that the self-tolerance system was destroyed. In the photosynthesis system,chlorophyll a increased and chlorophyll b decreased, which maintained the absorption of moisture and nutrients, as well as carbon assimilation(Li, H. S.etc.2004; Ashraf, M.etc.2007; JIA YB,etc.2008). The root was very sensitive to salt-alkaline stress,and all indexes showed the same trend of initial increase and subsequent decrease. The root superficial area, length, and root tip number showed the same trend of sharp decrease, suggesting that they were more sensitive to stress than others, but had some ability to reduce the harm from stress. In summary, the interactive effect of pH and salt content must be considered for growing triticale on salt-alkaline land. For the seedlings to have more tolerance, the seeding density should be increased and appropriate irrigation should be used to ensure the growth of triticale. Abbreviations SOD:superoxide dismutase; POD: peroxidase; CAT: catalase; MDA:malondialdehyde; ROS:reactive oxygen species; As A:ascorbic acid; GSH:glutathione. Declarations Ethics approval and consent to participate Ethics and Consent to Participate declarations: not applicable Clinical Trial Number Clinical trial number: not applicable Consent for publication All the listed authors have carefully reviewed and approved this manuscript.We all agree to submit it to BMC Plant Biology for publication and are aware of and accept the journal's publication policies. Availability of data and materials The authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by the Sericultural Industry Technical System of Shandong Province (Grant no. SDAIT-18-03), Quality Inspection and test Center for Sericulture Industry and Product of Ministry of Agriculture and Rural Affairs (Yantai) ,Shandong Engineering Research Center of Functional Crop Germplasm Innovation and Caltivation Utilization, Yantai Key Laboratory for Evaluation and Utilization of Silkworm Functional Substances. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yu Xiu, Rao Fu, Shuhui Ma, Qitong Huang, Lifeng Hua, Lei Nie, Huifen Liu and Zhenzu Li. The first draft of the manuscript was written by Yu Xiu and Zhenzu Li, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The authors confirm that the data and materials supporting the findings of this study are available within the article. 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International of Innovation and Applied Studies, 3: 511-516. Al-Khateeb, S. A.(2006). Effect of salinity and temperature on germination, growth and ion relations of Panicum turgidum Forssk. Bioresource Technology, 97:292-298. Dkhil, B.B.,& Denden, M.(2010). Salt stress induced changes in germination, sugars, starch and enzyme of carbohydrate metabolism in Abelmoschus esculentus(L.) Moench seeds. African Journal of Agricultural Research,5: 408-415. Yang, C.W., Shi, D. C., & Wang, D. L.(2008). Comparative effects of salt and alkali stresses on growth, osmotic adjustment and ionic balance of an alkali-resistant halophyte Suaeda glauca(Bge.). Plant Growth Regulation,56:179-190. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 22 Jul, 2025 Reviewers agreed at journal 18 Jul, 2025 Reviewers invited by journal 17 Jul, 2025 Editor assigned by journal 17 Jul, 2025 Editor invited by journal 16 Jul, 2025 Submission checks completed at journal 16 Jul, 2025 First submitted to journal 15 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7025898","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487684888,"identity":"98866754-6511-476e-b66d-6e1ea235f3dd","order_by":0,"name":"Yu Xiu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Xiu","suffix":""},{"id":487684890,"identity":"c285532b-863a-4c5a-b0b7-204ab581169a","order_by":1,"name":"Rao Fu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Rao","middleName":"","lastName":"Fu","suffix":""},{"id":487684894,"identity":"6d28fb10-3522-4137-bc7d-e42716eb4a98","order_by":2,"name":"Shuhui Ma","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shuhui","middleName":"","lastName":"Ma","suffix":""},{"id":487684895,"identity":"bcadb6e7-a338-4344-be7e-82a72d5c0089","order_by":3,"name":"Qitong Huang","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qitong","middleName":"","lastName":"Huang","suffix":""},{"id":487684897,"identity":"a99b06a0-8b95-4f7f-934e-37ee41b3c40b","order_by":4,"name":"Lifeng Hua","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lifeng","middleName":"","lastName":"Hua","suffix":""},{"id":487684899,"identity":"87eb4481-4f10-4827-b321-d0e656eb33d6","order_by":5,"name":"Lei Nie","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Nie","suffix":""},{"id":487684901,"identity":"44173efb-51b5-4c0a-8359-20d86a8c85ed","order_by":6,"name":"Huifen Liu","email":"","orcid":"","institution":"Shandong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Huifen","middleName":"","lastName":"Liu","suffix":""},{"id":487684904,"identity":"788049e2-4d35-47df-9fdf-805077377063","order_by":7,"name":"Zhenzu Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBACPiA+8MHgnxwbe/sB4rSwMTAwPpxRcMCYj+dMAtFamI15PhxInCfhYECkFv7jzyR4DO6kt0kwJDD8qNhGjC0H0iQkDJ7ltkk3HmDsOXObCC2MDcckDAyYc9tkDiQwM7YRowWoTCLBgDmdDUQSp4WNmdnggMHhBBK08LAxPmwwSDNsAwbyQaL8ws9//MHhP39s5OXb2w8++FFBhBYUcIBE9aNgFIyCUTAKcAEA2iY4ma1vF1MAAAAASUVORK5CYII=","orcid":"","institution":"Shandong Polytechnic","correspondingAuthor":true,"prefix":"","firstName":"Zhenzu","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-02 06:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7025898/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7025898/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87218268,"identity":"55e46824-b75c-484b-a298-caabc74384aa","added_by":"auto","created_at":"2025-07-21 15:49:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":341280,"visible":true,"origin":"","legend":"\u003cp\u003eGermination percentage, germination potential, germination index, length of germ, length of radicle in triticale seedlings in each group. Different letters indicate a significant difference (P≤0.01) among the treatments. CK the control(0 mmol·L-1 salt-saline concentration)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7025898/v1/635b9d800bf09c1957500330.png"},{"id":87218267,"identity":"806b577e-2181-4c2e-bcf7-c99843065fce","added_by":"auto","created_at":"2025-07-21 15:49:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":134167,"visible":true,"origin":"","legend":"\u003cp\u003eHeight of plant and wet weight of plant in triticale seedlings in each group, Different letters indicate a significant difference (P≤0.01) among the treatments. CK the control(0 mmol·L\u003csup\u003e-1\u003c/sup\u003e salt-saline concentration)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7025898/v1/58186c22837acdd60c08154e.png"},{"id":87219166,"identity":"8d40e7a2-4bbe-4eac-88b7-054d251f6db9","added_by":"auto","created_at":"2025-07-21 15:57:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":215900,"visible":true,"origin":"","legend":"\u003cp\u003eActivities of different antioxidant enzyme, SOD, POD and CAT in triticale seedlings in each group. Different letters indicate a significant difference (P≤0.01) among the treatments. CK the control without salt-saline treatment.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7025898/v1/d91dc6b21e094a4020bdd33a.png"},{"id":87217818,"identity":"6211cfc5-50f2-4a4a-b422-96fb5cfd069a","added_by":"auto","created_at":"2025-07-21 15:41:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67089,"visible":true,"origin":"","legend":"\u003cp\u003eProtein concentration in triticale seedlings in each group. Different letters indicate a significant difference (P≤0.01) among the treatments.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7025898/v1/7314617158d56feb849ead96.png"},{"id":87218269,"identity":"eb601f5f-34f5-4e4c-b814-efb7e1fa2f93","added_by":"auto","created_at":"2025-07-21 15:49:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":347824,"visible":true,"origin":"","legend":"\u003cp\u003eRoot structure parameters, as superficial area, length, volume, average diameter, number of tip of root in triticale seedlings in each group. Different letters indicate a significant difference (P≤0.01) among the treatments.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7025898/v1/b0dadf4d1e9a0a8a42a3d3af.png"},{"id":87217821,"identity":"a562193b-b315-448b-bb42-9d1a37b6a3a2","added_by":"auto","created_at":"2025-07-21 15:41:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":191421,"visible":true,"origin":"","legend":"\u003cp\u003ePhotosynthesis parameters, Cha, Chb and Cha/b concentration in triticale seedlings in each group. Different letters indicate a significant difference (P≤0.01) among the treatments.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7025898/v1/d9f3a1d82473ed40d8ca7e4e.png"},{"id":87219892,"identity":"c1815870-d68e-4cf4-a529-ed13c588cee1","added_by":"auto","created_at":"2025-07-21 16:07:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1777373,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7025898/v1/16191120-6053-47e2-b195-3ea7afb08b1c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Saline-Alkaline Stress on Seed Germination and Seedling Growth of Triticale","fulltext":[{"header":"Background","content":"\u003cp\u003eThe triticale with blue, purple-black, black, etc., grain color is a new variety of wheat, which has the advantages of turning green quickly, drought resistance, etc. The protein content is high and can reach 17.1–20.5% (Zhang long Yu, etc.). The content of essential amino acids such as isoleucine, leucine, etc., is 33.3–75.0% higher than the white wheat(Rong Li,etc.1995). Moreover, the contents of trace elements such as selenium, iodine, iron, etc., are higher than the white wheat(Yeo A R.1999; Ashraf M,2005; Ashraf M.2009). The anthocyanin content is also high(Xiao lan Li,etc.), and has active ingredients such as flavone, polysaccharides, etc., which have various pharmacological effects, such as free radical elimination, anti-aging, blood glucose reduction, etc(ZILIC S,etc.2011;BABBAR N,etc.2011;WAGNER J R,etc.1992). Hence, triticale is a health product with high nutritive value and wide prospective market applications(Zhang long Yu, etc.2016;Zhang long Yu,etc.2020;Yeo A R.1999).\u003c/p\u003e\u003cp\u003eThe saline-alkaline stress is one of the most important factors that affects the agriculture and land productivity. Moreover, 25% of global land area is affected by salinization(Ashraf M.2005;Ashraf M.2009; Asish Kumar Parida,etc.2005).The saline-alkaline area has increased at the speed of (1-1.5)×106 hm2 because of unreasonable irrigation, excessive use of chemical fertilizers, destruction of forests and vegetation, and global warming due to greenhouse effects, which have severely threatened the ecological environment and affected the sustainable development of agriculture(Darwish T, etc.2005; Lä uchli A,etc.2008). The northeast and northwest of China have saline-alkali land, comprising of alkaline salts (NaHCO\u003csub\u003e3\u003c/sub\u003e and Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e), and neutral salts (NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). The salinization and alkalization occurred simultaneously(Fen Fen Xu,etc.2012;Xiao Lei Zhang,etc.2013) producing salt and alkaline stress, and the alkaline salts have high pH, which could cause more serious harm to crops (Jian Xin Liu,etc.2016;Ya Ci Lv2021;Ya qi Chen,etc.2021). Therefore, salt stress and alkaline stress are research hotspots, but previous studies have always focused on either salt or alkaline stress, or comparison between them, and the combined saline-alkaline stress has been rarely studied.\u003c/p\u003e\u003cp\u003eThe influence of saline-alkaline stress on triticale germination and seedling growth stages has been rarely studied at home and abroad. Hence, we investigated the triticale SN-1 response to saline-alkaline stress during the germination and seedling growth stages to uncover the salt tolerance mechanism by determining the physiological indexes and active substance content changes, which provided a theoretical basis to uncover the molecular mechanism on the microcosmic level and the possibility of cultivating triticale on saline-alkaline land, leading to wide market application prospects for triticale.\u003c/p\u003e"},{"header":"Materials and Methods Experimental Design","content":"\u003cp\u003e\u003cb\u003e1. Mixed saline-alkaline condition simulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe chose two neutral salts (NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) and two alkaline salts (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and NaHCO\u003csub\u003e3\u003c/sub\u003e), mixed in different ratios(A: NaCl:Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 1:1, B: NaCl:NaHCO\u003csub\u003e3\u003c/sub\u003e 1:1, C:NaCl:Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e:NaHCO\u003csub\u003e3\u003c/sub\u003e:NaCO\u003csub\u003e3\u003c/sub\u003e1:1:1:1, D:Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e:NaCO\u003csub\u003e3\u003c/sub\u003e 1:1, E:NaHCO\u003csub\u003e3\u003c/sub\u003e:NaCO\u003csub\u003e3\u003c/sub\u003e 1:1), that were 25 different saline-alkaline conditions. We treated triticale SN-1 under these 25 conditions and tested seed germination and seedling stage physiological indexes.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2. River sand experiment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe experiment was conducted in the greenhouse provided by Shandong Silkworm Institute, Shandong, China, in 2021. The plastic pots used were 20cm in diameter and 30cm in height. They were filled with 300g river sand, and placed in the hydroponic box with Hoagland, to immerse the river sand with Hoagland. The nutrient solutionwas replaced after five days. The mode was irrigation and the experimental period was four weeks. We picked the plants with uniform growth for next period that was saline-alkaline stress.The hydroponic box was filled with 1500ml of each saline-alkaline solution, irrigated for one week, and then the triticale seedlings were used for testing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental Methods Stress treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor sowing triticale in late September 2021, first all seeds were disinfected with hypochlorite for 15 minutes, then rinsed with water until clean, and dried. The dried seeds were planted at a depth of 1- 2cm and a density of 15 seeds in the plastic pots with 300g river sand and then placed in the hydroponic box with all Hoagland.The selected 25 plastic pots were treated daily with about 1000 ml of 1∕2 Hoagland solution containing the above-described ratios of mixed saline-alkaline solutions to simulate natural saline-alkaline stress,while the same volume of nutrient solution without additional saline-alkaline solution was applied to the control group. All the pots were irrigated with nutrient solution and then treated by saline-alkaline stress for one week. Each saline-alkaline solution was added at 5:00–6:00pm daily for one week.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRecording of seed germination and survival\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe picked some triticale to test the germination, and made the above-mentioned 25 different ratios of saline-alkaline solutions and used them to simulate saline-alkaline conditions, the control group was treated with water, and every concentration had three repeats. The seeds were disinfected with hypochlorite for 15 minutes, rinsed with water until clean, and then placed on the petri dish (Φ = 7 cm). The petri dish had two layers of filter paper, with 30 seeds on the filter paper. The petri dishes were placed in a light incubator. The germination standard was when the length of the radicle was twice than that of the seed, and the length of the germ was equal to the seed (Hong wei Yang,etc.2017). We started counting the germinated seeds from the second day after sowing, and when the germination number remained the same for three days, it was regarded as the end of germination, The germination was continued for 10 days, and then the length of the radicle and germ were tested.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSeed germination index\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eGermination percentage = number of germinated seeds on the fifth day∕number of tested seeds× 100% (the germination number of every group remained the same five days later),Germination potential = number of germinated seeds on the second day∕number of tested seeds× 100% ( the daily germination number of the control group peaked on the second day)\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeasurement of seedling growth and other physiological parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOn the 35th day after sowing, the height and wet weight of three randomly selected seedlings were measured, and each measurement was repeated 3–5 times.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMechanisms of resistance in the seedling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUnder the saline-alkaline stress condition, the growth and development of plants are inhibited, and the plasma membrane is most seriously affected with the increase in concentration, the extravasation of electrolyte is increased, the free radical metabolism balance in the cell is destroyed(De zong Sui,etc.2007; Jing Tao.; Guo Qiang Wu,etc.2017), and the membrane lipid peroxides destroy the membrane system. malondialdehyde (MDA) was one of the production of the membrane lipids peroxide product, so the concentration of MDA increased that was the result of destruction of the cell membrane lipids and also the reason of destruction, the concentration of MDA reflected the cell membrane destruction degree and intensity of the reaction to the adversity condition (Jiu Ju Duan,etc.2006;Jun Guo Zhou,etc.2007;Chun mei Han,etc.2009). Plants produce large amounts of reactive oxygen species (ROS) due to saline-alkaline stress, because of resisting the destruction of the ROS, the plants established an effective ROS eliminating system during evolution, which could maintain the dynamic balance of ROS. The system comprises of an enzymatic antioxidant system and a non-enzymatic antioxidant system. The enzymatic antioxidant system comprises of SOD, CAT, POD, etc., while the non-enzymatic antioxidant system comprises of ascorbic acid (As A) and glutathione (GSH). The activity and concentration of antioxidant enzymes and the concentration of antioxidant material could reflect the elimination ability of ROS or intensity of resistance to adversity(Yan ru Guo,etc.2006).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeasurement of root length and other structural parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe root system has important influence on plants by absorbing and transporting water and nutrients in the soil and synthesizing and storing nutrition (Parida A K,etc.2005). The root system distribution affects the absorbance of nutrients and water from the soil, as well as the growth and developing ecology function setted up aboveground.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhotosynthesis parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePhotosynthesis is one of the most important physiological processes for plant growth and development(Munns R,etc.2008). Photosynthesis pigments facilitate the plant light reaction(Petrov V V,etc.2014),and chlorophyll is the main photosynthesis pigment (Ru La Sa,etc.2014).Saline-alkaline stress can destroy the photosynthesis system in a dose-response manner.\u003c/p\u003e\u003cp\u003eFresh leaves were collected, washed with distilled water, dried, shredded and mixed well.Thereafter, 0.2g of the leaves were weighed, added to 25ml calibration tubes, and treated with 10ml of 95% ethanol. The tubes were capped and placed in 60–80 ℃ water for extracting chlorophyll, until the green color of the leaves had faded. The tubes were cooled and 95% ethanol was added to scale, and the supernatant was tested for chlorophyll content.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll the presented data were the mean values of three replicates and expressed as mean ± SD. The data were analyzed using the SPSS 21 .0 software. The level for significant difference was p\u0026lt;0 .05 .\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSeed germination process and final germination percentage of triticale\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUnder saline-alkaline stress, seed germination is the key index for successful breeding of resistant varieties(Xu Zhao, etc).Generally, with increasing salt concentration, the water potential around the seeds decreases and the difference in water potential between the interior and exterior of the seed also decreases, limiting water absorption by the seeds.Insufficient water absorption impairs the utilization of the material stored in the endosperm and inhibits respiration, limiting seed germination, and results in a decreased final germination percentage and delay in seed germination. This study showed that saline-alkaline stress significantly affected the mean time of germination, germination percentage, germination potential and germination index .With the increase in salt-alkali concentration, the germination percentage, germination potential and germination index of the same treatment group decreased. When the alkaline stress increased, the reduction of germination parameters was maximum. When the concentration was 150mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the germination percentage, germination potential and germination index of the treatment group C were 60%, 46% and 20% ,respectively.When the concentration reached 200mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the germination percentage, germination potential and germination index declined to 20%, 57% and 36% ,respectively. The germination percentage of the treatment group C declined to 9% at 150mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e compared to 100mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the treatment group D declined to 49%, the germination potential of the treatment group C declined to 18% at 150mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e compared to 100mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and the treatment group D declined to 63%. It was observed that alkalinity seriously affected the germination percentage and germination potential, and seriously limited merging compared to the neutral salt. When the concentration reached 300 mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, every treatment group except group B had no merging, and the treatment groups D and E showed similar changes.\u003c/p\u003e\u003cp\u003eWith the increase in alkali concentration, the height of the gemmule and radicle of the same treatment group decreased. When the concentration reached 100mol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the height of the gemmule of the treatment group C was 63.14 mm, and when the concentration reached 150mmol\u0026middot; L \u003csup\u003e- \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, declined by 68%, the height of gemmule of the treatment group C declined by 9% of 100 mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Compared to 50mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the treatment group D declined by 66% ; when the concentration reached 50mmol\u0026middot; L \u003csup\u003e- \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the height of radicle of the treatment group C was 25.91mm. When the concentration reached 100mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the height of the radicle of the treatment group C declined by 63% of 100 mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Compared to 50mmol\u0026middot;L\u003csup\u003e- \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the treatment group D declined by 83%. Hence, alkalinity seriously affected thegemmule and radicle compared to neutral salt. When the concentration reached 300mmol\u0026middot;L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the gemmule did not merge except in the treatment groups A and B, and only the radicle of the treatment group A merged. The height of the radicle in the treatment groups D and E showed the same change.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSeedling growth and other physiological parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA previous study showed that the interactive effect of pH and salt significantly inhibited the \u003cb\u003eheight\u003c/b\u003e and dry weight of the entire plant, and that of roots, stems, and leaves, and the differences between any two groups were significant (Kai li Liu,etc.2005). Our study showed that height and wet weight of the plants in the treatment groups C, D and E rapidly decreased when the saline-alkaline concentration increased.This phenomenon was obvious in the treatment group E, and the wet weight and height of the plant were significantly affected by pH but not salt concentration .Hence, it can be concluded that when subjected to saline-alkaline stress, alkalinity was the major factor, and the wet weight and height of the plant decreased obviously with the increase in pH, which was significant in the treatment group E.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtective enzyme activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSaline-alkaline stress leads to extensive ROS production in plants that increases membrane permeability and lipid peroxidation, as well as leakage of electrolytes, which are harmful to the cytoplasmic membrane of plants(Dian Wang,etc.2012; Schreiber U,etc.1986).Plants secrete organic and inorganic substances to maintain their metabolic balance and synthesize protective enzymes to scavenge ROS, which enables the plant to tolerate saline-alkaline stress or reduce the damage caused by ROS(Liang Q,etc.2007). In this study, when triticale were cultured in 50mmol\u0026middot;L\u003csup\u003e- \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e salt-alkali concentration, the activities of SOD, POD and CAT increased compared to the control group. The antioxidant enzyme activity decreased significantly under high pH and salt concentration in every treatment group, and this trend was more remarkable under high pH, suggesting that pH was the main influencing factor compared to salt concentration, which implied that the plants had limited tolerance when challenged with saline-alkaline stress, and if the concentration exceeded the plant \u0026rsquo;s endurance limit, the long-term effects of ROS could inhibit the growth and survival of plants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSoluble protein content\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe concentration of soluble proteins showed a different trend compared to antioxidant enzyme activity.With increase in pH, the concentration of proteins increased and subsequently decreased, implying that under salt stress, more enzymes could be produced by plants to reduce the harm to plants. However, accumulation of ROS could damage the plants, which affected antioxidant enzyme production after the salt concentration was increased and then decreased.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRoot structure parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhen subjected to saline-alkaline stress, the morphological and physiological characteristics of the plants changed. Root formation of plants is based on hereditary characteristics, and restricted by soil ecological environment. In adverse situations, the root could change its form and distribution to adapt to harmful environment. A large study indicated that salt stress could limit root elongation and side root development, which can reduce root length, superficial area, and volume. Our study showed that when the salt concentration increased, the root superficial area, length, volume, and root tip number decreased, but not the average root diameter. With the increase in salinity, all parameters showed the same trend of initial increase followed by decrease, indicating that plants had certain tolerance which reduced the harm caused by salt. Among all the treatment groups, the treatment group E with the highest pH had the lowest values of all indexes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhotosynthesis parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo protect itself from lack of moisture absorption and light damage caused by the saline-alkaline stress, the plant reduces light absorption and improves its energy conversion efficiency to ensure carbon assimilation, keeping the leaf temperature relatively low.Chlorophyll a is the core pigment for photosynthesis that transforms solar energy into photochemical energy. Therefore, its content reflects the level of light energy conversion efficiency. Chlorophyll b is a light-harvesting pigment that is responsible for light absorption and transmission. Thus, regulation of the contents of chlorophyll a and b, as well as the chlorophyll a∕b ratio, is an important regulatory mechanism for resistance in the plant. In this study, chlorophyll a showed an increasing trend with the increase in saline-alkaline content, whereas chlorophyll b content decreased, which resulted in a higher chlorophyll a∕b ratio than that in the control group. These results indicated that saline-alkaline stress increased the core photosynthetic pigment but reduced the light-harvesting pigment,thus ensuring normal photosynthesis in triticale.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn order to examine the influence of salt-alkaline stress on triticale during the period of merging and seedling, we selected triticale SN-1 as the study subject for investigating the effect of salt-alkaline stress on the root growth during germination in river sand so that that the root can be easily washed. We started sowing in late September 2021 in a greenhouse in Shandong Silkworm Institute. The entire process was continued for one month and one week. In the beginning,irrigating nutrient solution was used for one month. Thereafter, different ratios of salt-alkaline solutions were added for one week(Mao Q Z,2008; Lynch J P.1995; Yi L P,etc.2006). At the trefoil stage, we collected the triticale blade, and detected the physiological and biochemical indexes to investigate the effects on SN-1. The results showed that during the period of merging, when pH and salt concentration were low, the germination percentage and potential were increased, the interactive effect of pH and salt concentration was more significant, which resulted in rapid decline in germination percentage.Therefore, low salt concentration could promote plant merging, the plant had some resistance to stress, but this resistance disappeared when the concentration was increased(Tong H,etc.2012; Saveyn A,etc.2010). The treatment groups D and E showed similar changing trends, indicating that high pH was the main influencer.The lengths of the germ and radicle decreased with the increase in salt-alkali concentration. When the pH was high, the radicle was more sensitive than the germ(Chu Wu,etc.2004). During the period of seedling, the height and wet weight of the plant showed similar trend, but unremarkable compared to the germ and radicle(Guo Cong Xu,etc.2016; Khan, M.A.etc.2003; Ahan, J.etc.2009), which indicated that seedling resistance was stronger than seeds in the merging period; activity of the protective enzymes SOD, POD and CAT increased when content was low(Zhang, X. Q.etc.2013). When pH and salt concentration were increased, the activity decreased, suggesting that at the beginning of stress, plants had some tolerance, which gradually decreased and eventually disappeared. The soluble protein content increased and subsequently decreased with the increase in salt-alkali content, indicating that the self-tolerance system was destroyed. In the photosynthesis system,chlorophyll a increased and chlorophyll b decreased, which maintained the absorption of moisture and nutrients, as well as carbon assimilation(Li, H. S.etc.2004; Ashraf, M.etc.2007; JIA YB,etc.2008). The root was very sensitive to salt-alkaline stress,and all indexes showed the same trend of initial increase and subsequent decrease. The root superficial area, length, and root tip number showed the same trend of sharp decrease, suggesting that they were more sensitive to stress than others, but had some ability to reduce the harm from stress. In summary, the interactive effect of pH and salt content must be considered for growing triticale on salt-alkaline land. For the seedlings to have more tolerance, the seeding density should be increased and appropriate irrigation should be used to ensure the growth of triticale.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSOD:superoxide dismutase;\u003c/p\u003e\n\u003cp\u003ePOD: peroxidase;\u003c/p\u003e\n\u003cp\u003eCAT: catalase;\u003c/p\u003e\n\u003cp\u003eMDA:malondialdehyde;\u003c/p\u003e\n\u003cp\u003eROS:reactive oxygen species;\u003c/p\u003e\n\u003cp\u003eAs A:ascorbic acid;\u003c/p\u003e\n\u003cp\u003eGSH:glutathione.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics and Consent to Participate declarations: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eClinical Trial Number\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the listed authors have carefully reviewed and approved this manuscript.We all agree to submit it to BMC Plant Biology for publication and are aware of and accept the journal\u0026apos;s publication policies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;the \u0026nbsp;Sericultural\u0026nbsp;Industry\u0026nbsp;Technical \u0026nbsp;System\u0026nbsp;\u0026nbsp;of\u0026nbsp;Shandong Province (Grant no. SDAIT-18-03),\u0026nbsp;Quality Inspection and test Center for Sericulture Industry and Product of Ministry of Agriculture and Rural Affairs (Yantai) ,Shandong\u0026nbsp;Engineering Research Center\u0026nbsp;of Functional \u0026nbsp;Crop\u0026nbsp;Germplasm \u0026nbsp;Innovation and Caltivation\u0026nbsp;Utilization,\u0026nbsp;Yantai \u0026nbsp;Key\u0026nbsp;\u0026nbsp;Laboratory\u0026nbsp;\u0026nbsp;for\u0026nbsp;\u0026nbsp;Evaluation \u0026nbsp; and \u0026nbsp; Utilization \u0026nbsp; of \u0026nbsp;Silkworm \u0026nbsp; Functional\u0026nbsp;Substances.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by\u0026nbsp;Yu Xiu,\u0026nbsp;Rao Fu,\u0026nbsp;\u0026nbsp;Shuhui Ma,\u0026nbsp;Qitong Huang, Lifeng Hua, Lei Nie, Huifen Liu\u0026nbsp;and\u0026nbsp;Zhenzu Li. The first draft of the manuscript was written by\u0026nbsp;Yu Xiu and Zhenzu Li,\u0026nbsp;and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data and materials supporting the findings of this study are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang long Yu, Rui Liu, Yu Song, etc. The nutrition quality analysis of the triticale of Yun series[J]\u003c/li\u003e\n\u003cli\u003eRong Li, Xin ping Li(1995), Positive developing the hard grain by the xinjiang resource advantage[J]. Xinjiang Agriculture Science,6:14-15.\u003c/li\u003e\n\u003cli\u003eYeo A R(1999). Predicting the interaction between the effects of salinity and climate change on crop plants[J]. 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Plant Growth Regulation,56:179-190.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"black wheat, mixed saline-alkaline stress, seed germination, seedling growth, activity of protective enzymes","lastPublishedDoi":"10.21203/rs.3.rs-7025898/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7025898/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo investigate the adaptation ability of triticale to saline-alkaline stress, SN-1 triticale was used in this study to determine the roles of different saline-alkaline ratio stress treatments during seed germination to seedling stage. In this study, we used two neutral salts (NaCl and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) and two alkaline salts (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and NaHCO\u003csub\u003e3\u003c/sub\u003e), mixed in 25 different ratios, We treated SN-1 under these 25 different saline-alkaline conditions and tested seed germination and seedling stage physiological indexes including germination percentage, germination energy, germination index, seedling stem length, seedling root length, the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), soluble proteins, etc. The results showed that during the period of merging, when pH and salt concentration were low, germination percentage and potential were increased. The treatment groups showed similar changing trends. The length of the germ and radicle decreased when salt-alkali concentration and especially the pH were increased, and the radicle was more sensitive than the germ. The activity of protective enzymes SOD, POD and CAT increased when salt-alkali content was low. Soluble protein content initially increased and then decreased when salt-alkali content increased. In photosynthesis system, Chlorophyll a increased while Chlorophyll b decreased. The root was very sensitive to salt-alkaline stress, all indexes showed the same trend of initial increase and subsequent decrease. The root superficial area, length, root tip number showed the same trend and decreased rapidly. All the physiological indexes indicated that the interactive effect of pH and salt concentration must be considered before sowing triticale in salt-alkaline land.\u003c/p\u003e","manuscriptTitle":"Effect of Saline-Alkaline Stress on Seed Germination and Seedling Growth of Triticale","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-21 15:41:51","doi":"10.21203/rs.3.rs-7025898/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-07-22T08:57:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276084615101675889283459458970568823519","date":"2025-07-19T03:19:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-17T07:57:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-17T07:52:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-16T06:50:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-16T05:14:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-07-16T03:15:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2cb2a3b0-4eae-4183-8377-44bec049bd03","owner":[],"postedDate":"July 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-21T15:41:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-21 15:41:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7025898","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7025898","identity":"rs-7025898","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}