Effect of in Vitro Salinity Stress on Growth Parameters, Osmo-protection and Oxidative Stress of Dianthus barbatus L. Calli

Document Type : Research Article

Authors

• Abedin Moshashaei ¹
• Mohammad Mehdi Jowkar ²

¹ , Kermanshah Branch, Islamic Azad University, Kermanshah, Iran

² Kermanshah Branch, Islamic Azad University, Kermanshah, Iran

https://doi.org/10.22067/jhorts4.v33i4.80302

Abstract

Introduction: Dianthus barbatus L. from Caryophyllaceae is an important herbaceous landscape ornamental plant in the temperate regions throughout the world. Although this ornamental plant is used as bedding plant, it also has other extensive applications such as cut flower and pot plant. Besides, in some countries the leaves of Dianthus barbatus are used for treatment of digestive disorders in their traditional medicine. Landscape and its quality, plays an essential role in human life. Nowadays, maintenance and development of urban landscape in many regions such as Iran is facing major challenging issues. Soil and water salinity are the most important challenges of urban landscape. Considering the challenges which landscape development faces in many parts of the country and the importance of this plant in landscape, the effect of salinity on D. barbatus during in vitro condition was studied in order to obtain some basic physiological information for future stress studies on this plant.
Materials and Methods: In vitro sterile plants were initially obtained from seeds by culturing them on basal MS medium. Calli were obtained by culturing leaf, stem and root explants on previously reported callogenesis optimization medium which was MS medium supplemented with 2µM BAP + 6µM NAA. Calli proliferation was then conducted on the same media. Proliferated calli with same size were subjected to MS medium supplemented with the mentioned plant growth regulators and various NaCl concentrations (0, 1, 3, 6, 9, 12 gl-1) in order to induce salinity stress. Various physiological aspects and stress related mechanisms such as growth, osmo-protection and oxidative stress mechanisms were consequently studied in the stressed Dianthus barbatus calli. Studied growth features were calli growth, final growth rate, fresh and dry weight. Total protein content, glycine-betaine content, and the activity of betaine aldehyde dehydrogenase (BAD) were studied as a part of osmo-protection mechanism. On the other hand, the activity of free radical scavenger enzymes such as sccccuperoxide dismutase (SOD), catalase (CAT), peroxidase (POX) and malondialdehyde damage biomarker content were studied in stressed calli as a part of oxidative stress mechanism. Total protein was measured using Bradford method. Glycine-betaine content was measured using spectroscopy method by KI-I2 reaction agent. The activity of betaine aldehyde dehydrogenase was evaluated using spectroscopy method by reduction with NAD+. SOD activity was measured using spectroscopy method and formation as an indicator. CAT activity was measured using spectroscopy method by measuring hydrogen peroxide breakdown. POX activity was measured using spectroscopy method by nitro-blue tetrazolium reduction activity. Malondialdehyde content was also measured by using spectroscopy method.
Results and Discussion: Results indicated that by increasing salinity level, calli growth and consequently fresh and dry weight reduced. Calli growth was dramatically reduced as salinity level reached above 1 gl-1. This slow growth was also observed at high stress levels such as 12 gl-1. By salinity increment, total protein content of the stressed calli significantly reduced. On the other hand, BAD activity increased as a result of salinity stress and consequently glycine-betaine content also increased significantly. The highest level of glycine-betaine content and also the activity of BAD activity were observed in 12gl-1 NaCl treated calli. A significant correlation between glycine-betaine content and BAD activity was observed. The activity of free radical scavenger enzymes such as SOD, CAT, POX also significantly increased by salinity stress increment. The highest activity of the mentioned free radical scavenger enzymes were observed in 12 gl-1 NaCl treated calli. CAT had the most activity among the mentioned studied free radical scavenger enzymes. The highest increase in POX activity was half of the control and also half of the activity increment of other studied free radical scavenger enzymes. Although the activity of free radical scavenger enzymes increased, the level of malondialdehyde damage biomarker also increased as the result of salinity stress increment. The highest amount of malondialdehyde content was seen in the 12gl-1 NaCl treated calli. A significant correlation between malondialdehyde content and the activity of free radical scavenger enzymes was observed.
Conclusion: In general, the calli of Dianthus barbatus cultured in vitro are sensitive to salinity stress induced by more than 1 gl-1 NaCl; indicating the stress tolerance threshold. The tolerance and resistance mechanisms performed well under stress condition and the stress calli resisted to salinity up to 12 gl-1 NaCl and survived without chlorosis.

Keywords

• Betaine aldehyde dehydrogenase
• Catalase, Glycine-betaine
• Malondialdehyde
• Superoxide dismutase

20.1001.1.20084730.1398.33.3.15.5

References

- Aeby H. 1984. Catalase in vitro. Methods Enzymology 105: 121–126.

2- Ahmadpour Dehkordi S., and Balouchi H.R. 2012. Effect of seed priming on antioxidant enzymes and lipids peroxidation of cell membrane in Black cumin (Nigella sativa) seedling under salinity and drought stress. Electronic Journal of Crop Production 5(4): 63-85. (In Persian with English Abstract)

3- Ali A., Afrasiab H., Naz S., Rauf M., and Iqhbal J. 2008. An efficient protocol for in vitro propagation of carnation (Dianthus caryophyllus L.). Pakistan Journal of Botany 40: 111-121.

4- Al-Khayre K.M., and Al-Bahranny A.M. 2002. Growth, water Contene, and proline accumulation in drought stressed callus of date palm. Biological Plantrum 48(1): 105-108.

5- Arakawa K., Katayama M., and Takabe T. 1990. Levels of Betaine and Betaine Aldehyde Dehydrogenase Activity in the Green Leaves, and Etiolated Leaves and Roots of Barley. Plant Cell Physiology, 31(6):797-803.

6- Arakawa K., Takabe T., Sugiyama T., and Akazawa T. 1987. Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody. Journal of Biochemistry 101: 1485-1488.

7- Ashraf M., and Foolad M.R. 2007. Roles of glycinebetaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany 59: 206-216.

8- Ashraf, M., and Harris, P.J.C. 2004. Potential Biochemical Indicators of Salinity Tolerance in Plants. Plant Science 166: 3-16.

9- Ayse S., and Alikamanoglu S. 2013. Antioxidant enzyme activities, malondialdehyde, and total phenolic content of PEG-induced hyperhydric leaves in sugar beet tissue culture. In Vitro Cell Developmental Biology Plant 49: 396-404.

10- Azizi M., Chehrazi M., and Zahedi S.M. 2011. Effects of Salinity Stress on Germination and Early Growth of Sweet William (Dianthus barbatus). Asian Journal of Agricultural Sciences 3(6): 453-458.

11- Boominathan R., and Doran P.M. 2002. Ni induced oxidative stress in roots of the Ni hyperaccumlator, Alyssum bertoloni. New Phytologist 156: 202-205.

12- Bradford M.M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anals of Biochemistry 72: 248-254.

13- Cai X., Niu G., Starman T. and Hall C. 2014. Response of six garden roses (Rosa× hybrida L.) to salt stress. Scientia Horticulturae 168: 27-32.

14- Chandra S., and Rawat D.S. 2015. Medicinal plants of the family Caryophyllaceae: a review of ethno-medicinal uses and pharmacological properties. Integrative Medicine Research 4: 123-131.

15- Compton M.E. 1994. Statistical method suitable for the analysis of plant tissue culture data. Plant Cell Tissue 37: 217- 242.

16- Dash M., and Panda S.K. 2001. Salt stress induced changes in growth and enzyme activities in germination Phaseolus mungo seeds. Biologia Plantarum 44(4): 587-589.

17- de Azevedo-Neto A.D., Prisco J.T., Eneas-Filho J., de Abreu C.E.B., and Gomes-Filho E. 2006. Effect of salt stress on antioxidative enzymes and lipid peroxidation in leaves and roots of salt-tolerant and salt-sensitive maize genotypes. Environmental and Experimental Botany 56(1): 87-94.

18- Doel J.M., and Wilkins H.F. 1999. Floriculture: principles and species. Prentic-Hall, Inc. New Jersy, USA. 613p.

19- Ebrahimi M., Zamani G.R., and Alizadeh Z. 2017. A study on the effects of water deficit on physiological and yield-related traits of pot marigold (Calendula officinalis L.). Iranian Journal of Medicinal and Aromatic Plants 33(3): 492-508. (In Persian with English Abstract)

20- FAO. 2010. Extent and causes of salt-affected soils in participating countries. Available on URL: http://www.fao.org/ag/AGL/agll/spuch/topic4.htm.

21- Galbally J., and Galbally E. 1997. Carnations and Pinks for garden and greenhouse. Timber Press, Portland Oregon, USA. 310p.

22- Ghasemi Ghasareh M. and Kafi M. 2015. Floriculture: Princiles and Practicals. Elm Afarin Publication, Esfahan, Iran. 313p.

23- Ghorbanli M., Ahmadi F., Monfared A., and Bakhshi Khaniki G.H. 2012. Effect of salt stress and its interaction with ascorbate on catalase, ascorbate peroxidase activity, proline and malondialdehyde in Cuminum cyminum L. four weeks after germination. Iranian Journal of Medicinal and Aromatic Plants 28(1): 14-27. (In Persian with English Abstract)

24- Gill S.S., and Tuteja N. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry 48(12): 909‐930.

25- Giri J. 2011. Glycinebetaine and abiotic stress tolerance in plants. Plant Signaling and Behavior 6(11):1746-1751.

26- Grieve C.M., and Grattan S.R. 1983. Rapid assay for determination of water soluble quaternary ammonium compounds. Plant Soil 70: 303-307.

27- Gunes A., Inal A., Alpaslan M., Eraslan F., Bagci E.G., and Cicek N. 2007. Salicylic acid induced changes on some physiological parameters symptomatic for oxidative stress and mineral nutrition in maize (Zea mays L.) grown under salinity. Journal of Plant Physiology 164: 728-736.

28- Haouala F., and Jaziri F. 2009. In Vitro Propagation of Carnation (Dianthus Caryophyllus L.) Under Salt Stress. Pakistan Journa of Biotechnology 6(1-2): 27-30.

29- Haouala F., Hannachi C., and Zid E. 2003. Exploitation de la variabilite soma-clonale pour la recherche d’oeillet (Dianthus caryophyllus L.) tolerant à la salinite. Tropicultura 21(1): 16-21.

30- Harati E., Kashefi B., and Matinizadeh M. 2017. Investigation of Reducing Detrimantal Effects of Salt Stress on Morphological and Physiological Traits of (Thymus daenensis Celak.) through Salicylic Acid Application. Plant Production Technology 8(2): 111-125. (In Persian with English Abstract)

31- Hashemi Esfahani S.A. 2008. Modern Floriculture Extension. Sepehr Publication, Tehran, Iran. 192 p. (In Persian)

32- ICARDA. 2002. International cooperation Highlands regional program. Available on: URL: http// www.icarda.cgiar.org.

33- Irma T., Jolan C., Gabriella S., Ferenc H., Attila P., Gabriella K., Agnes S., Margit S., and Laszlo E. 2002. Acclimation of tomato plants to salinity stress after a salicylic acid pre- treatment. Acta Biologica Szegediensis 46(3-4): 55-56.

34- Kar M. and Mishra D. 1976. Catalase, peroxidase and polyphenol oxidase activities during rice leaf senescence. Plant Physiology, 578:315-319.

35- Li-Ping B., Fang-Gong S., Ti-Da G., Zhao-Hui S., Yin-Yan L., and Guang-Sheng Z. 2006. Effect of soil drought stress on leaf water status, membrane permeability and enzymatic antioxidant system of maize. Pedosphere 16: 326-332.

36- Lutts S., Almansoure M., Kinet J.M. 2004. Salinity and water stree have contrasting effects on the relation ship between growth and cell viability during and after stress stress exposure in durum wheat callus. Plant Science 167: 9-18.

37- Ma X., Zheng J., Zhang X., Hu Q., and Qian R. 2017. Salicylic Acid Alleviates the Adverse Effects of Salt Stress on Dianthus superbus (Caryophyllaceae) by Activating Photosynthesis, Protecting Morphological Structure, and Enhancing the Antioxidant System. Frontiers in Plant Science 8: 600

38-Makela P., Peltonen-Sainio P., Jokinen K., Pehu E., Setala H., Hinkkanen R., and Somersalo S. 1996. Uptake and translocation of foliar applied glycinebetaine in crop plants. Plant Science 121: 221-230.

39- McCord J.M., and Fridovich I. 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). Journal of Biological Chemistry 244(22):6049-6055.

40- Minami M., and Yoshikawa H. 1979. A simplified assay method of superoxide dismutase activity for clinical use. Clinical Chemistry Acta 92: 337-342.

41- Miri H.R., and Moghadam A.Z. 2015. The Effect of External Usage of Glycine Betaine on Corn (Zea mays L.) in Drought Condition. Iranian Journal of Field Crops Research 12(4): 704-717. (In Persian with English Abstract)

42- Moshashaei A., Jowkar M.M., and Farshadfar E. 2019. Callogenesis optimization and the effect of in vitro osmotic stress on Dianthus barbatus (L.) calli. Journal of Plant Process and Function 7(27): 237-252. (In Persian with English Abstract)

43- Munir N., and Aftab F. 2009. The role of polyethylene glycol (PEG) pretreatment in improving sugarcane’s salt (NaCl) tolerance. Turkish Journal of Botany 33(6): 407-415.

44- Munns R. 2002. Comparative physiology of salt and water stress. Plant, Cell and Environment 25: 239-250.

45- Nisreen Y., El-Kichaoui A., and Ayesh B. 2015. Enhancement of Salinity Tolerance in (Dianthus caryophyllus L.) using in vitro Selection Technique and Isolation of Potential Salinity Tolerance. Islamic University- Gaza Deanship of Postgraduate Studies, Faculty of Science, Department of Biological Sciences. pp: 67-87.

46- Orcutt D.M., and Nilsen E.T. 2000. The physiology of plants under stress, soil and biotic factors. John Wiley and Sons. New York, USA. pp: 177-235.

47- Oz Aydin S., Dirmenci T., Tumen G., and Can Baser K.H. 2006. Plants used as analgesic in the folk medicine of Turkey. Proceedings of the 4th International Congress of Ethnobotany (ICEB 2005). pp: 167-171.

48- Parida A.K., and Das A.B. 2005. Salt tolerance and salinity effects on plants: a review. Ecotoxicology and Environmental Safety 60: 324-349.

49- Patade V.Y., Lokhande V.H., and Suprasanna P. 2014. Exogenous application of proline alleviates salt induced oxidative stress more efficiently than glycine betaine in sugarcane cultured cells. Sugar Technology, 16 (1): 22-29.

50- Razavizadeh R., and Ehsanpour A. 2004. Studying the tolerance of Medicago sativa L. calli to drought stress under UV-C irridation. Iranian Biology Journal 17(2): 1-12. (In Persian with English Abstract)

51- Rostami M., and Mazaheri S. 2010. Effect of salinity stress on Dianthus barbatus seed germination. Medicinal Plant National Conference. Sari, Iran. (In Persian)

52- Sakthivelu G., Akitha M.K., Giridhar P., Rajasekaran T., Ravishankar G.A., Nedev T., and Kosturkova A. 2008. Drought- induced alteration in growth, osmotic potential and in vitro regeneration of Soybean cultivars. Plant Physiology 34(1-2): 103-112.

53- Salmalian M., Ghasemnejad A., and Mashayekhi K. 2017. The study of quantitative and qualitative changes of Stevia rebaudiana Bertoni. Callus under the influence of salicylic acid and salt in solid and liquid culture conditions. Quarterly Journal of Ecophytochemisrty of Medicinal Plants 19(3): 23-39. (In Persian with English Abstract)

54- Sotiropoulos T.E. 2007. Effect of NaCl and CaCl2 on growth and contents of minerals, chlorophyll, proline and sugars in the apple rootstock M4 cultured in vitro. Biologia Plantarum 51: 177-180.

55- Soundararajan P., Abinaya M., Yoo G., Park M., Sowbiya M., and Byoung R.J. 2015. Silicon Alleviates Salt stress by Modulating Antioxidant Enzyme Activities in Dianthus caryophyllus ‘Tula’. Environmental Biotechnology 56(2): 233-239.

56- Sudhakar C., Lakshmi A., and Giridarakumar S. 2001. Changes in the antioxidant enzyme efficacy in two high yielding genotypes of mulberry (Morus alba L.) under NaCl salinity. Plant Science 141: 613-619.

57- Sunita D., and Gupta A.K. 2002. Tissue culture studies in carnation (Dianthus caryophyllus L.). MS Thesis. College of Agriculture. CCS Haryana Agricultural University, Hisar, India. pp: 68-96.

58- Takahashi R., Liu S., and Takano T. 2009. Isolation and Characterization of Plasma membrane Na+/H+ antiporter Genes from Salt-Sensitive and Salt- Tolerant (Sweet William). Journal of Plant Physiology 166: 301- 309.

59- Tehranifar A., and Kharrazi M. 2016. In Vitro Culture of Carnation (Dianthus caryophyllus L.) Focusing on the Problem of Vitrification. Journal of Biodiversity and Environmental Sciences 5(13): 1-6.

60- Thakur M., and Sharma A.D. 2005. Salt-stress-induced proline accumulation in germinating embryos: evidence suggesting a role of proline in seed germination. Journal of Arid Environments 62: 517–523.

61- Varier A., Vari A.K., and Dadlani M. 2010. The subcellular basis of seed priming. Current Science 99(4): 450-456.

62- Yildirim O., Aras S., and Ergul A. 2004. Response of Antioxidant System to Short-term NaCl stress in Grapevine Root stock-1616c and Vitis vinifera L. cv. Razaki. Acta Biological Cracoviensia 46: 151-158.

63- Zhang F.Q., Zhang H.X., Wang G.P., Xu L.L., and Shen Z.G. 2009. Cadmium induced accumulation of hydrogen peroxide in the leaf apoplast of Phaseolus aureusand Vicia sativaand the roles of different antioxidant enzymes. Journal of Hazardous Materials 168(1): 76-84.