Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 139, Issue 3, pp 531–546 | Cite as

NaCl-induced stress: physiological responses of six halophyte species in in vitro and in vivo culture

  • Yuping Xiong
  • Hanzhi Liang
  • Haifeng Yan
  • Beiyi Guo
  • Meiyun Niu
  • Shuangyan Chen
  • Shuguang Jian
  • Hai Ren
  • Xinhua Zhang
  • Yuan Li
  • Songjun Zeng
  • Kunlin Wu
  • Feng Zheng
  • Jaime A. Teixeira da Silva
  • Guohua MaEmail author
Original Article


To investigate the mechanisms underlying salt tolerance, physiological parameters of six halophyte species [Vitex rotundifolia L., Clerodendrum inerme (L.) Gaertn, Phyla nodiflora (L.) Greene, Scaevola sericea Vahl, Alternanthera bettzickiana (Regel) Nichols, and Dracaena cambodiana Pierre ex Gagn] under NaCl stress in in vitro and in vivo culture tests were examined. Membership function analysis and cluster analysis divided the six species, based on their salt tolerance level, into three groups: Group 1 (highly salt tolerant) included C. inerme, A. bettzickiana and S. sericea; Group 2 (moderately salt tolerant) included P. nodiflora; Group 3 (weakly salt tolerant) included V. rotundifolia and D. cambodiana. In response to in vitro NaCl stress, all six species showed a significant increase in the activities of antioxidant enzymes. NaCl stress enhanced free proline content in the leaves of all six species. CAT, SOD activity and proline accumulation were significantly correlated with the growth of C. inerme, P. nodiflora and A. bettzickiana under in vitro NaCl treatment. We conclude that NaCl-tolerant plants may suffer slight damage within a certain salt concentration, as evidenced by the activities of antioxidant enzymes and the accumulation of free proline.

Key message

Six halophytic species showed a different salt tolerance level under in vitro and in vivo culture tests, due to the activities of antioxidant enzymes and the accumulation of free proline.


Halophyte Clerodendrum inerme (L.) Gaertn Vitex rotundifolia L. Phyla nodiflora (L.) Greene Scaevola sericea Vahl Alternanthera bettzickiana (Regel) Nichols Dracaena cambodiana Pierre ex Gagn Salt tolerance Physiological parameters 







Fresh weight


Indole-3-butyric acid




Murashige and Skoog


α-Naphthaleneacetic acid




Superoxide dismutase


Root length


Rooting percentage


Visible injury



The authors thank Guangdong Zhongke Qilin Landscape Co., Ltd. for providing D. cambodiana plantlets.

Author contributions

YPX and HZL prepared samples for all analyses. HFY, SYC, BYG and MYN conducted the statistical analysis of physiological changes. YPX and HFY were also involved in statistical analysis and wrote the manuscript. JATdS offered interpretative analysis and co-wrote the manuscript. SGJ, HR, XHZ, YL, SJZ, KLW, FZ, JATdS and GHM designed the experiment and provided guidance for the study. All authors read and approved the manuscript.


This work was financially supported by the National Key Research and Development Program of China (2016YFC1403000/2016YFC1403002), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA13020500) and the National Science and Technology Support Program (2015BAL04B04). The funding agencies had no role in the design, analysis, and interpretation of the data or writing of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


  1. AbdElgawad H, Zinta G, Hegab MM, Pandey R, Asard H, Abuelsoud W (2016) High salinity induces different oxidative stress and antioxidant responses in maize seedlings organs. Front Plant Sci 7:276. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Abogadallah GM (2010) Antioxidative defense under salt stress. Plant Signal Behav 5:369–374CrossRefGoogle Scholar
  3. Ahanger MA, Tomar NS, Tittal M, Argal S, Agarwal RM (2017) Plant growth under water/salt stress: ROS production; antioxidants and significance of added potassium under such conditions. Physiol Mol Biol Plants 23:731–744. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Ahmad N, Javed SB, Khan MI, Anis M (2013) Rapid plant regeneration and analysis of genetic fidelity in micropropagated plants of Vitex trifolia: an important medicinal plant. Acta Physiol Plant 35:2493–2500. CrossRefGoogle Scholar
  5. Ahmed ABA, Pallela R, Rao AS, Rao MV, Mat Taha R (2011) Optimized conditions for callus induction, plant regeneration and alkaloids accumulation in stem and shoot tip explants of Phyla nodiflora. Span J Agric Res 9:1262. CrossRefGoogle Scholar
  6. Al Hassan M, Pacurar A, López-Gresa MP, Donat-Torres MP, Llinares JV, Boscaiu M, Vicente O (2016) Effects of salt stress on three ecologically distinct Plantago species. PLoS ONE 11:e0160236. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Ali A, Iqbal N, Ali F, Afzal B (2012) Alternanthera bettzickiana (Regel) G. Nicholson, a potential halophytic ornamental plant: growth and physiological adaptations. Flora 207:318–321. CrossRefGoogle Scholar
  8. Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207. CrossRefGoogle Scholar
  9. Bendaly A, Messedi D, Smaoui A, Ksouri R, Bouchereau A, Abdelly C (2016) Physiological and leaf metabolome changes in the xerohalophyte species Atriplex halimus induced by salinity. Plant Physiol Biochem 103:208–218. CrossRefPubMedGoogle Scholar
  10. Cha-um S, Somsueb S, Samphumphuang T, Kirdmanee C (2013) Salt tolerant screening in eucalypt genotypes (Eucalyptus spp.) using photosynthetic abilities, proline accumulation, and growth characteristics as effective indices. In Vitro Cell Dev Biol 49:611–619. CrossRefGoogle Scholar
  11. Chen X, Min D, Yasir TA, Hu Y-G (2012) Evaluation of 14 morphological, yield-related and physiological traits as indicators of drought tolerance in Chinese winter bread wheat revealed by analysis of the membership function value of drought tolerance (MFVD). Field Crop Res 137:195–201. CrossRefGoogle Scholar
  12. Domingo C, Lalanne E, Catalá MM, Pla E, Reig-Valiente JL, Talón M (2016) Physiological basis and transcriptional profiling of three salt-tolerant mutant lines of rice. Front Plant Sci 7:1462. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Duan H, Ma Y, Liu R, Li Q, Yang Y, Song J (2018) Effect of combined waterlogging and salinity stresses on euhalophyte Suaeda glauca. Plant Physiol Biochem 127:231–237. CrossRefPubMedGoogle Scholar
  14. Gao J, Li J, Luo C, Yin L, Li S, Yang G, He G (2011) Callus induction and plant regeneration in Alternanthera philoxeroides. Mol Biol Rep 38:1413–1417. CrossRefPubMedGoogle Scholar
  15. Gao W, He M, Liu J, Ma X, Zhang Y, Dai S, Zhou Y (2018) Overexpression of Chrysanthemum lavandulifolium ClCBF1 in Chrysanthemum morifolium ‘White Snow’ improves the level of salinity and drought tolerance. Plant Physiol Biochem 124:50–58. CrossRefPubMedGoogle Scholar
  16. Ghars MA, Parre E, Debez A, Bordenave M, Richard L, Leport L, Bouchereau A, Savouré A, Abdelly C (2008) Comparative salt tolerance analysis between Arabidopsis thaliana and Thellungiella halophila, with special emphasis on K+/Na+ selectivity and proline accumulation. J Plant Physiol 165:588–599. CrossRefPubMedGoogle Scholar
  17. Gregorio GB, Senadhira D, Mendoza RD (1997) Screening rice for salinity tolerance. IRRI discussion paper series NO. 22. International Rice Research Instituete, Manila, PhilippinesGoogle Scholar
  18. Hajiboland R, Bahrami-Rad S, Akhani H, Poschenrieder C (2018) Salt tolerance mechanisms in three Irano-Turanian Brassicaceae halophytes relatives of Arabidopsis thaliana. J Plant Res 131:1029–1046. CrossRefPubMedGoogle Scholar
  19. He S, Han Y, Wang Y, Zhai H, Liu Q (2008) In vitro selection and identification of sweet potato (Ipomoea batatas (L.) Lam.) plants tolerant to NaCl. Plant Cell Tissue Organ Cult 96:69. CrossRefGoogle Scholar
  20. Hu L, Li H, Chen L, Lou Y, Amombo E, Fu J (2015) RNA-seq for gene identification and transcript profiling in relation to root growth of bermudagrass (Cynodon dactylon) under salinity stress. BMC Genomics 16:575. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hu G, Liu Y, Duo T, Zhao B, Cui G, Ji J, Kuang X, Ervin EH, Zhang X (2018) Antioxidant metabolism variation associated with alkali-salt tolerance in thirty switchgrass (Panicum virgatum) lines. PLoS ONE 13:e0199681. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Jaarsma R, de Vries RS, de Boer AH (2013) Effect of salt stress on growth, Na+ accumulation and proline metabolism in potato (Solanum tuberosum) cultivars. PLoS ONE 8:e60183. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Khan MA, Qaiser M (2006) Halophytes of Pakistan: characteristics, distribution and potential economic usages. In: Khan MA, Böer B, Kust GS, Barth H-J (eds) Sabkha ecosystems, vol II. West and Central Asia. Springer, Dordrecht, pp 129–153. CrossRefGoogle Scholar
  24. Kumari A, Das P, Parida AK, Agarwal PK (2015) Proteomics, metabolomics, and ionomics perspectives of salinity tolerance in halophytes. Front Plant Sci 6:537. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Liang W, Ma X, Wan P, Liu L (2018) Plant salt-tolerance mechanism: a review. Biochem Biophys Res Commun 495:286–291. CrossRefPubMedGoogle Scholar
  26. Liu Q, Tang J, Wang W, Zhang Y, Yuan H, Huang S (2018) Transcriptome analysis reveals complex response of the medicinal/ornamental halophyte Iris halophila Pall. to high environmental salinity. Ecotoxicol Environ Saf 165:250–260. CrossRefPubMedGoogle Scholar
  27. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498. CrossRefPubMedGoogle Scholar
  28. Mu D, Zwiazek JJ, Li Z, Zhang W (2016) Genotypic variation in salt tolerance of Ulmus pumila plants obtained by shoot micropropagation. Acta Physiol Plant 38:188. CrossRefGoogle Scholar
  29. Muchate NS, Nikalje GC, Rajurkar NS, Suprasanna P, Nikam TD (2016) Plant salt stress: adaptive responses, tolerance mechanism and bioengineering for salt tolerance. Bot Rev 82:371–406. CrossRefGoogle Scholar
  30. Murashige T, Skoog F (1962) A reviced medium for rapid growth and bioassays with tobacoo tissue culture. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  31. Nataraj M, Kher MM, Teixeira da Silva JA (2016) Micropropagation of Clerodendrum L. species: a review. Rend Fis Acc Lincei 27:169–179. CrossRefGoogle Scholar
  32. Nikalje GC, Mirajkar SJ, Nikam TD, Suprasanna P (2018) Multifarious role of ROS in halophytes: signaling and defense. In: Zargar SM, Zargar MY (eds) Abiotic stress-mediated sensing and signaling in plants: an omics perspective. Springer, Singapore, pp 207–223CrossRefGoogle Scholar
  33. Parida AK, Jha B (2010) Salt tolerance mechanisms in mangroves: a review. Trees 24:199–217CrossRefGoogle Scholar
  34. Prerostova S, Dobrev PI, Gaudinova A, Hosek P, Soudek P, Knirsch V, Vankova R (2017) Hormonal dynamics during salt stress responses of salt-sensitive Arabidopsis thaliana and salt-tolerant Thellungiella salsuginea. Plant Sci 264:188–198. CrossRefPubMedGoogle Scholar
  35. Ravikiran KT, Krishnamurthy SL, Warraich AS, Sharma PC (2018) Diversity and haplotypes of rice genotypes for seedling stage salinity tolerance analyzed through morpho-physiological and SSR markers. Field Crops Res 220:10–18. CrossRefGoogle Scholar
  36. Rossatto T, do Amaral MN, Benitez LC, Vighi IL, Braga EJB, de Magalhães Júnior AM, Maia MAC, da Silva Pinto L (2017) Gene expression and activity of antioxidant enzymes in rice plants, cv. BRS AG, under saline stress. Physiol Mol Biol Plants 23:865–875. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Saadia M, Jamil A, Akram NA, Ashraf M (2012) A study of proline metabolism in canola (Brassica napus L.) seedlings under salt stress. Molecules 17:5803–5815. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Saini S, Kaur N, Pati PK (2018) Reactive oxygen species dynamics in roots of salt sensitive and salt tolerant cultivars of rice. Anal Biochem 550:99–108. CrossRefPubMedGoogle Scholar
  39. Sarabi B, Bolandnazar S, Ghaderi N, Ghashghaie J (2017) Genotypic differences in physiological and biochemical responses to salinity stress in melon (Cucumis melo L.) plants: prospects for selection of salt tolerant landraces. Plant Physiol Biochem 119:294–311. CrossRefPubMedGoogle Scholar
  40. Silva-Ortega CO, Ochoa-Alfaro AE, Reyes-Agüero JA, Aguado-Santacruz GA, Jiménez-Bremont JF (2008) Salt stress increases the expression of p5cs gene and induces proline accumulation in cactus pear. Plant Physiol Biochem 46:82–92. CrossRefPubMedGoogle Scholar
  41. Soares ALC, Geilfus CM, Carpentier SC (2018) Genotype-specific growth and proteomic responses of maize toward salt stress. Front Plant Sci 9:661. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Tang J, Liu Q, Yuan H, Zhang Y, Wang W, Huang S (2018) Molecular cloning and characterization of a novel salt-specific responsive WRKY transcription factor gene IlWRKY2 from the halophyte Iris lactea var. chinensis. Genes Genomics 40:893–903. CrossRefPubMedGoogle Scholar
  43. Wang YH, Bhalla PL (2004) Somatic embryogenesis from leaf explants of Australian fan flower, Scaevola aemula R. Br. Plant Cell Rep 22:408–414. CrossRefPubMedGoogle Scholar
  44. Wang H, Wang J, Wu X, Wu XP, Shen HY, Luo Y, Dai HF, Mei WL (2015a) HPLC-ESI-MS analysis of flavonoids obtained from tissue culture of Dracaena cambodiana. Chem Res Chin Univ 31:38. CrossRefGoogle Scholar
  45. Wang J, Meng Y, Li B, Ma X, Lai Y, Si E, Yang K, Xu X, Shang X, Wang H, Wang D (2015b) Physiological and proteomic analyses of salt stress response in the halophyte Halogeton glomeratus. Plant, Cell Environ 38:655–669. CrossRefGoogle Scholar
  46. Wang B, Guo X, Zhao P, Ruan M, Yu X, Zou L, Yang Y, Li X, Deng D, Xiao J, Xiao Y, Hu C, Wang X, Wang X, Wang W, Peng M (2017) Molecular diversity analysis, drought related marker-traits association mapping and discovery of excellent alleles for 100-day old plants by EST-SSRs in cassava germplasms (Manihot esculenta Cranz). PLoS ONE 12:e0177456. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Xie Y, Luo H, Hu L, Sun X, Lou Y, Fu J (2014) Classification of genetic variation for cadmium tolerance in Bermudagrass [Cynodon dactylon (L.) Pers.] using physiological traits and molecular markers. Ecotoxicology 23:1030–1043. CrossRefPubMedGoogle Scholar
  48. Yao L, Wang J, Li B, Meng Y, Ma X, Si E, Ren P, Yang K, Shang X, Wang H (2018) Transcriptome sequencing and comparative analysis of differentially-expressed isoforms in the roots of Halogeton glomeratus under salt stress. Gene 646:159–168. CrossRefPubMedGoogle Scholar
  49. Yi X, Sun Y, Yang Q, Guo A, Chang L, Wang D, Tong Z, Jin X, Wang L, Yu J, Jin W, Xie Y, Wang X (2014) Quantitative proteomics of Sesuvium portulacastrum leaves revealed that ion transportation by V-ATPase and sugar accumulation in chloroplast played crucial roles in halophyte salt tolerance. J Proteom 99:84–100. CrossRefGoogle Scholar
  50. Younis A, Riaz A, Ikram S, Nawaz T, Hameed M, Fatima S, Batool R, Ahmad F (2013) Salinity-induced structural and functional changes in 3 cultivars of Alternanthera bettzickiana (Regel) G. Nicholson. Turk J Agric For 37:674–687. CrossRefGoogle Scholar
  51. Yuan F, Lyu MJ, Leng BY, Zheng GY, Feng ZT, Li PH, Zhu XG, Wang BS (2015) Comparative transcriptome analysis of developmental stages of the Limonium bicolor leaf generates insights into salt gland differentiation. Plant, Cell Environ 38:1637–1657. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Yuping Xiong
    • 1
    • 2
  • Hanzhi Liang
    • 1
    • 2
  • Haifeng Yan
    • 3
  • Beiyi Guo
    • 1
    • 2
  • Meiyun Niu
    • 1
    • 2
  • Shuangyan Chen
    • 1
  • Shuguang Jian
    • 1
  • Hai Ren
    • 1
  • Xinhua Zhang
    • 1
  • Yuan Li
    • 1
  • Songjun Zeng
    • 1
  • Kunlin Wu
    • 1
  • Feng Zheng
    • 1
  • Jaime A. Teixeira da Silva
    • 4
  • Guohua Ma
    • 1
    Email author
  1. 1.Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical GardenThe Chinese Academy of SciencesGuangzhouChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.Cash Crop Institute of Guangxi Academy of Agricultural SciencesNanningChina
  4. 4.Miki-choJapan

Personalised recommendations