Skip to main content
SpringerLink
Log in

Toward Unravelling the Genetic Determinism of the Acquisition of Salt and Osmotic Stress Tolerance Through In Vitro Selection in Medicago truncatula

  • Protocol
  • First Online:
Book cover Functional Genomics in Medicago truncatula

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1822))

Abstract

Changes in global climate and the nonstop increase in demographic pressure have provoked a stronger demand for agronomic resources at a time where land suitable for agriculture is becoming a rare commodity. They have also generated a number of abiotic stresses which exacerbate effects of diseases and pests and result in physiological and metabolic disorders that ultimately impact on yield when and where it is most needed. Therefore, a major scientific and agronomic challenge today is that of understanding and countering the impact of stress on yield. In this respect, in vitro biotechnology would be an efficient and feasible breeding alternative, particularly now that the genetic and genomic tools needed to unravel the mechanisms underlying the acquisition of tolerance to stress have become available. Legumes in general play a central role in a sustainable agriculture due to their capacity to symbiotically fix the atmospheric nitrogen, thereby reducing the need for fertilizers. They also produce grains that are rich in protein and thus are important as food and feed. However, they also suffer from abiotic stresses in general and osmotic stress and salinity in particular. This chapter provides a detailed overview of the methods employed for in vitro selection in the model legume Medicago truncatula for the generation of novel germplasm capable of resisting NaCl- and PEG-induced osmotic stress. We also address the understanding of the genetic determinism in the acquisition of stress resistance, which differs between NaCl and PEG. Thus, the expression of genes linked to growth (WEE1), in vitro embryogenesis (SERK), salt tolerance (SOS1) proline synthesis (P5CS), and ploidy level and cell cycle (CCS52 and WEE1) was upregulated under NaCl stress, while under PEG treatment the expression of MtWEE1 and MtCCS52 was significantly increased, but no significant differences were observed in the expression of genes MtSERK1 and MtP5CS, and MtSOS1 was downregulated. A number of morphological and physiological traits relevant to the acquisition of stress resistance were also assessed, and methods used to do so are also detailed.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ochatt SJ (2015) Agroecological impact of an in vitro biotechnology approach of embryo development and seed filling in legumes. Agron Sustain Dev 35:535–552

    Article  CAS  Google Scholar 

  2. Atif RM, Patat-Ochatt EM, Svabova L et al (2013) Gene transfer in legumes. In: Lüttge U, Beyschlag W, Francis D, Cushman J (eds) Progress in botany, vol 74. Springer, Berlin, Heidelberg, pp 37–100

    Chapter  Google Scholar 

  3. Gatti I, Guindón F, Bermejo C et al (2016) In vitro tissue culture in breeding programs of leguminous pulses: use and current status. Plant Cell Tissue Organ Cult 127:543–559

    Article  CAS  Google Scholar 

  4. Jacob C, Carrasco B, Schwember AR (2016) Advances in breeding and biotechnology of legume crops. Plant Cell Tissue Organ Cult 127:561–584

    Article  CAS  Google Scholar 

  5. Araújo SS, Beebe S, Crespi M et al (2015) Abiotic stress responses in legumes: strategies used to cope with environmental challenges. Crit Rev Plant Sci 34:237–280

    Article  CAS  Google Scholar 

  6. Araújo S, Balestrazzi A, Faè M et al (2016) MtTdp2α-overexpression boosts the growth phase of Medicago truncatula cell suspension and increases the expression of key genes involved in the antioxidant response and genome stability. Plant Cell Tissue Organ Cult 127:675–680

    Article  CAS  Google Scholar 

  7. Duque AS, López-Gómez M, Kráčmarová J et al (2016) Genetic engineering of polyamine metabolism changes Medicago truncatula responses to water deficit. Plant Cell Tissue Organ Cult 127:681–690

    Article  CAS  Google Scholar 

  8. Elmaghrabi AM, Ochatt S, Rogers HJ, Francis D (2013) Enhanced tolerance to salinity following cellular acclimation to increasing NaCl levels in Medicago truncatula. Plant Cell Tissue Org Cult 114:61–70

    Article  CAS  Google Scholar 

  9. Elmaghrabi AM, Rogers HJ, Francis D, Ochatt SJ (2017) PEG induces high expression of the cell cycle checkpoint gene WEE1 in embryogenic callus of Medicago truncatula: potential link between cell cycle checkpoint regulation and osmotic stress. Front Plant Sci 8:1479. https://doi.org/10.3389/fpls.2017.01479

    Article  PubMed  PubMed Central  Google Scholar 

  10. Badri M, Ilahi H, Huguet T et al (2007) Quantitative and molecular genetic variation in sympatric populations of Medicago laciniata and M. truncatula (Fabaceae): relationships with eco-geographical factors. Genet Res 89:107–122

    Article  PubMed  CAS  Google Scholar 

  11. www.noble.org/medicago/ecotypes.html

  12. Motan JF, Becana M, Iturbeormaetxe I et al (1994) Drought induces oxidative stress in pea plants. Planta 194:346–352

    Google Scholar 

  13. Gonzalez EM, Aparicio-Tejo PM, Gondon AJ et al (1998) Water-deficit effects on carbon and nitrogen metabolism of pea nodules. J Exp Bot 49:1705–1714

    Article  CAS  Google Scholar 

  14. Costa França MG, Pham Thi AT, Pimentel C et al (2000) Differences in growth and water relations among Phaseolus vulgaris cultivars in response to induced drought stress. Environ Exp Bot 43:227–237

    Article  PubMed  Google Scholar 

  15. Galvez L, Gonzalez EM, Arrese-Igor C (2005) Evidence for carbon flux shortage and strong carbon/nitrogen interaction in pea nodules at early stages of water stress. J Exp Bot 56:2551–2561

    Article  PubMed  CAS  Google Scholar 

  16. Zahaf O, Blanchet S, de Zélicourt A et al (2012) Comparative transcriptomic analysis of salt adaptation in roots of contrasting Medicago truncatula genotypes. Mol Plant 5:1068–1081

    Article  CAS  PubMed  Google Scholar 

  17. Alcântara A, Morgado RS, Silvestre S et al (2015) A method to identify early-stage transgenic Medicago truncatula with improved physiological response to water deficit. Plant Cell Tissue Organ Cult 122:605–616

    Article  CAS  Google Scholar 

  18. Nunes CMJ, Araújo SS, Marques da Silva J et al (2008) Physiological responses of the legume model Medicago truncatula cv. Jemalong to water deficit. Environ Exp Bot 63:289–296

    Article  CAS  Google Scholar 

  19. Queiros F, Fidalgo F, Santos I, Salema R (2007) In vitro selection of salt tolerant cell lines in Solanum tuberosum L. Biol Plant 51:728–734

    Article  CAS  Google Scholar 

  20. Feki K, Quintero FJ, Pardo JM, Masmoudi K (2011) Regulation of durum wheat NaC/HC exchanger TdSOS1by phosphorylation. Plant Mol Biol 76:545–556

    Article  PubMed  CAS  Google Scholar 

  21. Tester M, Leigh RA (2001) Partitioning of transport processes in roots. J Exp Bot 52:442–457

    Article  Google Scholar 

  22. Tester M, Davenport R (2003) Na+ tolerance and Na+ transport in higher plants. Ann Bot 91:503–507

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Kim DY, Jin J-Y, Alejandro S et al (2010) Overexpression of AtABCG36 improves drought and salt stress resistance in Arabidopsis. Physiol Plant 139:170–180

    Article  PubMed  CAS  Google Scholar 

  24. Alet IA, Sánchez SH, Cuevas CJ et al (2012) New insights into the role of spermine in Arabidopsis thaliana under long-term salt stress. Plant Sci 182:94–100

    Article  PubMed  CAS  Google Scholar 

  25. Munns R (2002) Comparative physiology of salt and water stress. Plant Cell Environ 25:239–250

    Article  CAS  PubMed  Google Scholar 

  26. Hasegawa PM, Bressan RA, Zhu J-K, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51:463–499

    Article  PubMed  CAS  Google Scholar 

  27. Hernandez JA, Campillo A, Jimenez A et al (1999) Response of antioxidant system and leaf water relation to NaCl stress in pea plants. New Phytol 141:241–251

    Article  CAS  PubMed  Google Scholar 

  28. Abebe T, Guanzi AC, Martin B, Cushman JC (2003) Tolerance of mannitol accumulating transgenic wheat to water stress and salinity. Plant Physiol 131:1748–1755

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Trapp S, Feificova D, Rasmussen FN, Bauer-Gottein P (2008) Plant uptake of NaCl in relation to enzyme kinetics and toxic effects. Environ Exp Bot 64:1–7

    Article  CAS  Google Scholar 

  30. Trinchant JC, Boscari A, Spennato G et al (2004) Proline betaine accumulation and metabolism in alfalfa plants under sodium chloride stress. Exploring its compartmentalization in nodules. Plant Physiol 135:1583–1594

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Negrão S, Schmöckel SM, Tester M (2017) Evaluating physiological responses of plants to salinity stress. Ann Bot 119:1–11. https://doi.org/10.1093/aob/mcw191

    Article  PubMed  Google Scholar 

  32. Yang W-J, Rich PJ, Axtell JD et al (2003) Genotypic variation for glycinebetaine in sorghum. Crop Sci 43:162–169

    Article  CAS  Google Scholar 

  33. Parida AK, Das AB (2005) Salt tolerance and salinity effects on plants: a review. Ecotoxicol Environ Saf 60:324–349

    Article  PubMed  CAS  Google Scholar 

  34. Aydi S, Sassi S, Debouba M et al (2010) Resistance of Medicago truncatula to salt stress is related to glutamine synthetase activity and sodium sequestration. J Plant Nutr Soil Sci 173:892–899

    Article  CAS  Google Scholar 

  35. Surjus A, Durand M (1996) Lipid changes in soybean root membranes in response to salt treatment. J Exp Bot 47:17–23

    Article  CAS  Google Scholar 

  36. Chen J-B, Wang S-M, Jing R-L, Mao X-G (2009) Cloning the PvP5CS gene from common bean (Phaseolus vulgaris) and its expression patterns under abiotic stresses. J Plant Physiol 166:12–19

    Article  PubMed  CAS  Google Scholar 

  37. Choudhary NL, Sairam RK, Tyagi A (2005) Expression of 1-pyrroline−5 carboxylate synthetase gene during drought in rice (Oryza sativa L.). Indian J Biochem Biophys 42:366–370

    PubMed  CAS  Google Scholar 

  38. Silva-Ortega CO, Ochoa-Alfaro AE, Reyes-Agüero JA et al (2008) Salt stress increases the expression of p5cs gene and induces proline accumulation in cactus pear. Plant Physiol Biochem 46:82–92

    Article  PubMed  CAS  Google Scholar 

  39. Somboonwatthanaku I, Dorling S, Leung S et al (2010) Proline biosynthetic gene expression in tissue cultures of rice (Oryza sativa L.) in response to saline treatment. Plant Cell Tissue Organ Cult 103:369–376

    Article  CAS  Google Scholar 

  40. Sairam RK, Tyagi A (2004) Physiology and molecular biology of salinity stress tolerance in plants. Curr Sci 86:407–421

    CAS  Google Scholar 

  41. Vinocur B, Altman A (2005) Recent advances in Engineering plant tolerance to abiotic stress: achievements and limitations. Curr Opin Biotechnol 16:1913–1923

    Article  CAS  Google Scholar 

  42. Zsigmond L, Szepesib A, Tari I et al (2012) Overexpression of the mitochondrial PPR40 gene improves salt tolerance in Arabidopsis. Plant Sci 182:87–93

    Article  PubMed  CAS  Google Scholar 

  43. Shi HZ, Lee B-H, Wu S-J, Zhu J-K (2003) Overexpression of a plasma membrane Na?/H? Antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21:81–85

    Article  PubMed  CAS  Google Scholar 

  44. Türkan I, Demiral T (2009) Recent developments in understanding salinity tolerance. Environ Exp Bot 67:2–9

    Article  CAS  Google Scholar 

  45. Tang R-J, Liu H, Bao Y et al (2010) The woody plant poplar has a functionally conserved salt overly sensitive pathway in response to salinity stress. Plant Mol Biol 74:367–380

    Article  PubMed  CAS  Google Scholar 

  46. Li D, Zhang Y, Hu X et al (2011) Transcriptional profiling of Medicago truncatula under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses. BMC Plant Biol 11:109. https://doi.org/10.1186/1471-2229-11-109

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Cabuslay SG, Ito O, Alejar AA (2002) Physiological evaluation of responses of rice (Oryza sativa L.) to water deficit. Plant Sci 163:815–827

    Article  CAS  Google Scholar 

  48. Levitt J (1972) Responses of plants to environmental stresses. Academic Press, New York, NY

    Google Scholar 

  49. Zhu J-K (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:2273–2467

    Article  CAS  Google Scholar 

  50. Golldack D, Li C, Mohan H et al (2014) Tolerance to drought and salt stress in plants: unraveling the signaling networks. Front Plant Sci 5:151. https://doi.org/10.3389/fpls.2014.00151

    Article  PubMed  PubMed Central  Google Scholar 

  51. Valliyodan B, Nguyen HT (2006) Understanding regulatory networks and engineering for enhanced drought tolerance in plants. Curr Opin Plant Biol 9:189–195

    Article  PubMed  CAS  Google Scholar 

  52. Fulda S, Mikkat S, Stegmann H, Horn R (2011) Physiology and proteomics of drought stress acclimation in sunflower (Helianthus annuus L.). Plant Biol 13:632–642

    Article  PubMed  CAS  Google Scholar 

  53. Deinlein U, Stephan AB, Horie T et al (2014) Plant salt-tolerance mechanisms. Trends Plant Sci 19:371–379

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Ochatt S, Muilu R, Ribalta F (2008) Cell morphometry and osmolarity as early indicators of the onset of embryogenesis from cell suspension cultures of grain legumes and model systems. Plant Biosyst 142:480–486

    Article  Google Scholar 

  55. Ochatt SJ, Moessner A (2010) Rounding up plant cells. Int J Plant Biol 1:e8. https://doi.org/10.4081/pb.2010.e8

    Article  Google Scholar 

  56. Cushman JC, Bohnert HJ (2000) Genomic approaches to plant stress tolerance. Curr Opin Plant Biol 3:117–124

    Article  PubMed  CAS  Google Scholar 

  57. Sreenivasulu N, Varshney RK, Kavi Kishor PB et al (2004) Functional genomics for tolerance to abiotic stress in cereals: a functional genomics approach. In: Gupta PK, Varshney RK (eds) Cereal genomic. Springer, Dordrecht, pp 483–514. https://doi.org/10.1007/1-4020-2359-6_16

    Chapter  Google Scholar 

  58. De Schutter K, Joubes J, Cools T et al (2007) Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint. Plant Cell 19:211–225

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930

    Article  PubMed  CAS  Google Scholar 

  60. Zhao L, Wang P, Hou H et al (2014) Transcriptional regulation of cell cycle genes in response to abiotic stresses correlates with dynamic changes in histone modifications in maize. PLoS One 9:e106070. https://doi.org/10.1371/journal.pone.0106070

    Article  PubMed  PubMed Central  Google Scholar 

  61. Roy S (2016) Function of MYB domain transcription factors in abiotic stress and epigenetic control of stress response in plant genome. Plant Signal Behav 11:e1117723. https://doi.org/10.1080/15592324.2015.1117723

    Article  PubMed  CAS  Google Scholar 

  62. Sorrell DA, Marchbank A, McMahon K et al (2002) A WEE1 homologue from Arabidopsis thaliana. Planta 215:518–522

    Article  PubMed  CAS  Google Scholar 

  63. West G, Inzé D, Beemster GTS (2004) Cell cycle modulation in the response of the primary root of Arabidopsis to salt stress. Plant Physiol 135:1050–1058

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Skirycz A, Claeys H, De Bodt S et al (2011) Pause-and-stop: the effects of osmotic stress on cell proliferation during early leaf development in Arabidopsis and a role for ethylene signaling in cell cycle arrest. Plant Cell 23:1876–1888

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Gonzalez N, Hernould M, Delmas F et al (2004) Molecular characterization of a WEE1 gene homologue in tomato (Lycopersicon esculentum mill.). Plant Mol Biol 56:849–861

    Article  PubMed  CAS  Google Scholar 

  66. Gonzalez N, Gevaudant F, Hernould M, Chevalier C, Mouras A (2007) The cell cycle associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit. Plant J 51:642–655

    Article  PubMed  CAS  Google Scholar 

  67. Sun Y, Dilkes BP, Zhang C et al (1999) Characterization of maize (Zea mays L.) Wee1 and its activity in developing endosperm. Proc Natl Acad Sci U S A 96:4180–4185

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  68. Cebolla A, Vinardell JM, Kiss E et al (1999) The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants. EMBO J 18:4476–4484

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  69. Larson-Rabin Z, Li Z, Masson PH, Day CD (2009) FZR2/CCS52A1 expression is a determinant of endoreduplication and cell expansion in Arabidopsis. Plant Physiol 149:874–884

    Article  PubMed  PubMed Central  Google Scholar 

  70. Nolan KE, Rose RJ, Gorst JG (1989) Regeneration Medicago truncatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants. Plant Cell Rep 8:278–281

    Article  PubMed  CAS  Google Scholar 

  71. Chabaud M, Larsonneau C, Marmouget C et al (1996) Transformation of barrel medic (Medicago truncatula Gaertn.) by agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12 nodulin promoter fused to the gus reporter gene. Plant Cell Rep 15:305–310

    Article  PubMed  CAS  Google Scholar 

  72. Hoffmann B, Trinh TH, Leung J et al (1997) A new Medicago truncatula line with superior in vitro regeneration, transformation and symbiotic properties isolated through cell culture selection. Mol Plant-Microbe Interact 10:307–315

    Article  CAS  Google Scholar 

  73. Trinh TH, Ratet P, Kondorosi E et al (1998) Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. Falcata lines improved in somatic embryogenesis. Plant Cell Rep 17:345–355

    Article  CAS  PubMed  Google Scholar 

  74. Wang X-D, Nolan KE, Irwanto RR et al (2011) Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells. Ann Bot 107:599–609

    Article  PubMed  PubMed Central  Google Scholar 

  75. Iantcheva A, Vlahova M, Bakalova E et al (1999) Regeneration of diploid annual medics via direct somatic embryogenesis promoted by thidiazuron and benzylaminopurine. Plant Cell Rep 18:904–910

    Article  CAS  Google Scholar 

  76. Iantcheva A, Vlahova M, Trinh TH et al (2001) Assessment of polysomaty, embryo formation and regeneration in liquid media for various species of diploid annual Medicago. Plant Sci 160:621–627

    Article  PubMed  CAS  Google Scholar 

  77. Svetoslavova G, Vlahova M, Iantcheva A et al (2005) High frequency plant regeneration of diploid Medicago coerulea through somatic embryogenesis. Biotech Biotech Equip 19:57–61

    Article  Google Scholar 

  78. Duque AS, Pires AS, Santos DM et al (2006) Efficient somatic embryogenesis and plant regeneration from long-term cell suspension cultures of Medicago truncatula cv. Jemalong. In Vitro Cell Dev Biol Plant 42:270–273

    Article  CAS  Google Scholar 

  79. Ochatt S, Jacas L, Patat-Ochatt EM, Djennane S (2013) Flow cytometric analysis and molecular characterization of agrobacterium tumefaciens-mediated transformants of Medicago truncatula. Plant Cell Tissue Organ Cult 113:237–244

    Article  CAS  Google Scholar 

  80. Anjanasree K, Neelakandan WK (2012) Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications. Plant Cell Rep 31:597–620

    Article  CAS  Google Scholar 

  81. Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188

    Article  PubMed  CAS  Google Scholar 

  82. Jain SM (2001) Tissue culture-derived variation in crop improvement. Euphytica 118:153–166

    Article  CAS  Google Scholar 

  83. Sotirova V, Shtereva L, Zagorska N et al (1999) Resistance responses of plants regenerated from tomato anther and somatic tissue cultures to Clavibacter michiganense. Israel J Plant Sci 47:237–243

    Article  Google Scholar 

  84. Lu S, Peng X, Guo Z et al (2007) In vitro selection of salinity tolerant variants from triploid bermudagrass (Cynodon transvaalensis x C. dactylon) and their physiological responses to salt and drought stress. Plant Cell Rep 26:1413–1420

    Article  PubMed  CAS  Google Scholar 

  85. Ochatt SJ, Power JB (1989) Selection for salt/drought tolerance using protoplast and explant-derived tissue cultures of Colt cherry (Prunus avium x pseudocerasus). Tree Physiol 5:259–266

    Article  PubMed  CAS  Google Scholar 

  86. Ochatt SJ, Power JB (1989) Cell wall synthesis and salt (saline) sensitivity of Colt cherry (Prunus avium x pseudocerasus) protoplasts. Plant Cell Rep 8:365–367

    Article  PubMed  CAS  Google Scholar 

  87. Ochatt SJ, Marconi PL, Radice S et al (1999) In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plants. Plant Cell Tissue Org Cult 55:1–8

    Article  Google Scholar 

  88. Chen S, Chai M, Jia Y et al (2011) In vitro selection of salt tolerant variants following 60Co gamma irradiation of longterm callus cultures of Zoysia matrella [L.] Merr. Plant Cell Tissue Organ Cult 107:493–500

    Article  CAS  Google Scholar 

  89. Davenport SB, Gallego SM, Benavides MP et al (2003) Behaviour of antioxidant defense system in the adaptive response to salt stress in Helianthus annuus L. cells. Plant Growth Reg 40:81–88

    Article  CAS  Google Scholar 

  90. Gu R, Liu Q, Pei D, Jiang X (2004) Understanding saline and osmotic tolerance of Populus euphratica suspended cells. Plant Cell Tissue Organ Cult 78:261–265

    Article  CAS  Google Scholar 

  91. Basu S, Gangopadhyay G, Mukherjee BB, Gupta S (1997) Plant regeneration of salt adapted callus of indica rice (var.Basmati 370) in saline condition. Plant Cell Tissue Organ Cult 50:153–159

    Article  CAS  Google Scholar 

  92. Tao L, Van Staden SJ (2000) Selection and characterization of sodium chloride-tolerant callus of Glycine max (L) Merr cv. Acme. Plant Growth Reg 31:195–207

    Article  Google Scholar 

  93. Zhu J-K (2000) Genetic analysis of plant salt tolerance using Arabidopsis. Plant Physiol 124:941–948

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  94. Shi H, Quintero FJ, Pardo JM, Zhu JK (2002) The putative plasma membrane Na+/H+ antiporter SOS1 controls long-distance Na+ transport in plants. Plant Cell 14:465–477

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  95. Merchan F, Breda C, Hormaeche JP et al (2003) A Kruppel-like transcription factor gene is involved in salt stress responses in Medicago spp. Plant Soil 257:1–9

    Article  CAS  Google Scholar 

  96. Veatch ME, Smith SE, Vandemark G (2004) Shoot biomass production among accession of Medicago truncatula exposed to NaCl. Crop Sci 44:1008–1013

    Article  Google Scholar 

  97. Machuka J, Rasha AO, Magiri E et al (2008) In vitro selection and characterization of drought tolerant somaclones of tropical Maize (Zea mays L.). Biotechnology 7:641–650

    Article  Google Scholar 

  98. Claeys H, Van Landeghem S, Dubois M et al (2014) What is stress? Dose-response effects in commonly used in vitro stress assays. Plant Physiol 165:519–527

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  99. Zhang L, Ma H, Chen T et al (2014) Morphological and physiological responses of cotton (Gossypium hirsutum L.) plants to salinity. PLoS One 9(11):e112807

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  100. Kurth E, Cramer GR, Lauchli A, Epstein E (1986) Effects of NaCI and CaCl2 on cell enlargement and cell production in cotton roots. Plant Physiol 82:1102–1106

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  101. Farooq M, Wahid A, Kobayashi N (2009) Plant drought effects, mechanisms and management. Agron Sustain Dev 29:185–212

    Article  Google Scholar 

  102. Ochatt SJ (1994) In vitro selection for salt/drought tolerance in Colt cherry. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, Somaclonal variation in crop improvement II, vol 36. Springer-Verlag, Heidelberg, pp 223–238

    Google Scholar 

  103. Nolan KE, Irwanto RR, Rose RJ (2003) Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures. Plant Physiol 133:218–230

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  104. Nolan KE, Kurdyukov S, Rose RJ (2009) Expression of the somatic embryogenesis receptor like kinase1 (SERK1) gene is associated with developmental change in the life cycle of model legume Medicago truncatula. J Exp Bot 60:1759–1771

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  105. Liu L, White MJ, MacRae TH (1999) Transcription factors and their genes in higher plants. Functional domains, evolution and regulation. Eur J Biochem 262:247–257

    Article  PubMed  CAS  Google Scholar 

  106. Miki Y, Hashiba M, Hisajima S (2001) Establishment of salt stress tolerant rice plant through set up NaCl treatment in vitro. Plant Biol 44:391–395

    Article  Google Scholar 

  107. Yamaguchi T, Blumwald E (2005) Developing salt-tolerant crop plants: challenges and opportunities. Trends Plant Sci 10(12):615–620

    Article  PubMed  CAS  Google Scholar 

  108. Leone A, Costa A, Tucci M, Grillo S (1994) Adaptation versus shock response to polyethylene glycol-induced low water potential in cultured potato cells. Physiol Plant 92:21–30

    Article  CAS  Google Scholar 

  109. Gossett DR, Banks SW, Millhollon EP, Lucas MC (1996) Antioxidant response to NaCl stress in a control and a NaCl-tolerant cotton cell line grown in the presence of paraquat, buthionine sulfoximine and exogenous glutathione. Plant Physiol 112:803–809

    Article  PubMed  PubMed Central  Google Scholar 

  110. Shankhdhar D, Shankhdhar SC, Mani SC (2000) In vitro selection for salt tolerance in rice. Plant Biol 43:477–480

    Article  CAS  Google Scholar 

  111. Ochatt SJ, Pontecaille C, Rancillac M (2000) The growth regulators used for bud regeneration and shoot rooting affect the competence for flowering and seed set in regenerated plants of protein pea. In Vitro Cell Dev Biol Plants 36:188–193

    Article  CAS  Google Scholar 

  112. Rubio MC, González EM, Minchin FR et al (2002) Effects of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa overexpressing superoxide dismutases. Plant Physiol 115:531–540

    Article  CAS  Google Scholar 

  113. Hu H, Xiong L, Yang Y (2005) Rice SERK1 gene positively regulates somatic embryogenesis of cultured cell and host defense response against fungal infection. Planta 222:107–117

    Article  PubMed  CAS  Google Scholar 

  114. Sakthivelu G, Akitha Devi MK, Giridhar P et al (2008) Drought-induced alterations in growth, osmotic potential and in vitro regeneration of soybean cultivars. Gen Appl Plant Physiol 34:103–112

    CAS  Google Scholar 

  115. Guóth A, Benyo D, Csiszar J et al (2010) Relationship between osmotic stress-induced abscisic acid accumulation, biomass production and plant growth in drought-tolerant and -sensitive wheat cultivars. Acta Physiol Plant 32:719–727

    Article  CAS  Google Scholar 

  116. Mahmood I, Razzaq A, Hafiz AI et al (2012) Interaction of callus selection media and stress duration for in vitro selection of drought tolerant callus of wheat. Afr J Biotechnol 11:4000–4006

    CAS  Google Scholar 

  117. Rai MK, Kaliaa RK, Singh R et al (2011) Developing stress tolerant plants through in vitro selection—an overview of the recent progress. Environ Exp Bot 71:89–98

    Article  Google Scholar 

  118. Yang L, Li Y, Shen H (2012) Somatic embryogenesis and plant regeneration from immature zygotic embryo cultures of mountain ash (Sorbus pohuashanensis). Plant Cell Tissue Organ Cult 109:547–556

    Article  CAS  Google Scholar 

  119. Michel BE, Kaufmann MR (1973) The osmotic potential of polyethylene glycol 6000. Plant Physiol 51:914–916

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  120. Lu Z, Neumann PM (1998) Water-stressed maize, barley and rice seedlings show species diversity in mechanisms of leaf growth inhibition. J Exp Bot 49:1945–1952

    Article  CAS  Google Scholar 

  121. Srivastava DK, Gupta VK, Sharma DR (1995) In vitro selection and characterization of water stress tolerant callus cultures of tomato (Lycopersicon esculentum L.). Indian J Plant Physiol 38:99–104

    Google Scholar 

  122. Attree SM, Pomeroy MK, Fowke LC (1995) Development of white spruce (Picea glauca (Moench.) Voss) somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage. J Exp Bot 46:433–439

    Article  CAS  Google Scholar 

  123. Igasaki T, Sato T, Akashi N et al (2003) Somatic embryogenesis and plant regeneration from immature zygotic embryos of Cryptomeria japonica D. Don. Plant Cell Rep 22:239–243

    Article  PubMed  CAS  Google Scholar 

  124. Macovei A, Balestrazzi A, Confalonieri M, Carbonera D (2010) The Tyrosyl-DNA phosphodiesterase gene family in Medicago truncatula Gaertn.: bioinformatic investigation and expression profiles in response to copper- and PEG-mediated stress. Planta 232:393–407

    Article  PubMed  CAS  Google Scholar 

  125. Balestrazzi A, Confalonieri M, Macovei A, Carbonera D (2011) Seed imbibition in Medicago truncatula Gaertn.: expression profiles of DNA repair genes in relation to PEG-mediated stress. J Plant Physiol 168:706–713

    Article  PubMed  CAS  Google Scholar 

  126. Pintos B, Martin JP, Centeno ML et al (2002) Endogenous cytokinin levels in embryogenic and non- embryogenic calli of Medicago arborea L. Plant Sci 163:955–960

    Article  CAS  Google Scholar 

  127. Elmaghrabi AM, Ochatt SJ (2006) Isoenzymes and flow cytometry for the assessment of true-to-typeness of calluses and cell suspension of barrel medic prior to regeneration. Plant Cell Tissue Organ Cult 85:31–43

    Article  CAS  Google Scholar 

  128. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  129. Iantcheva A, Slavov S, Prinsen E et al (2005) Somatic embryogenesis of the model legume- Medicago truncatula and other diploid Medics. Plant Cell Tissue Organ Cult 81:37–43

    Article  CAS  Google Scholar 

  130. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  CAS  PubMed  Google Scholar 

  131. Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204

    Article  CAS  Google Scholar 

  132. das Neves L, Duque S, de Almeida J et al (1999) Repetitive somatic embryogenesis in Medicago truncatula ssp. Narbonensis and M. truncatula Gaertn cv. Jemalong. Plant Cell Rep 18:398–405

    Article  Google Scholar 

  133. Uchimiya T, Murashige M (1974) Evaluation of parameters in the isolation of viable protoplasts from cultured tobacco cells. Plant Physiol 54:936–944

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  134. Zafar Y, Nenz E, Damiani F et al (1995) Plant regeneration from explant and protoplast derived calluses of Medicago littoralis. Plant Cell Tissue Organ Cult 41:41–48

    Article  Google Scholar 

  135. Denchev P, Velcheva M, Atanassov A (1991) A new approach to direct somatic embryogenesis in Medicago. Plant Cell Rep 10:338–341

    Article  PubMed  CAS  Google Scholar 

  136. Vergana R, Verbc F, Pitto L et al (1990) Reversible variation in the methylation pattern of carrot DNA during somatic embryogenesis. Plant Cell Tissue Organ Cult 8:697–701

    Google Scholar 

  137. Scarpa GM, Pupilli F, Damiani F, Arcioni S (1993) Plant regeneration from callus and protoplasts in Medicago polymorpha. Plant Cell Tissue Organ Cult 35:49–57

    Article  CAS  Google Scholar 

  138. Chabaud M, de Carvalho-Niebel F, Barker DG (2003) Efficient transformation of Medicago truncatula cv. Jemalong using the hypervirulent Agrobacterium tumefaciens strain AGL1. Plant Cell Rep 22:46–51

    Article  PubMed  CAS  Google Scholar 

  139. Troll W, Lindsley J (1955) A photometric method for the determination of proline. J Biol Chem 215:655–660

    PubMed  CAS  Google Scholar 

  140. Boukel M, Houassine D (1997) Adaptation au stress hydrique de quelques variétés de blé dur (Triticum durum). Thèse Magistère, INA, Algérie 90 pp

    Google Scholar 

  141. Plummer DT (1987) Introduction to practical biochemistry, 3rd edn. McGraw Hill Book Company Ltd, London, pp 179–180

    Google Scholar 

  142. Yazici I, Türkan I, Sekmen SH et al (2007) Salinity tolerance of purslane (Portulaca oleracea L.) is achieved by enhanced antioxidative system, lower level of lipid peroxidation and proline accumulation. Environ Exp Bot 61:49–57

    Article  CAS  Google Scholar 

  143. Smart RE, Bingham GE (1974) Rapid estimates of relative water content. Plant Physiol 53:258–260

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  144. Ochatt SJ (2008) Flow cytometry in plant breeding. Cytometry A 73:581–598

    Article  PubMed  CAS  Google Scholar 

  145. Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47:189–194

    Article  PubMed  CAS  Google Scholar 

  146. Oparka KJ (1991) Uptake and compartmentation of fluorescent probes by plant cells. J Exp Bot 42:565–579

    Article  CAS  Google Scholar 

  147. Spadafora ND, Doonan JH, Herbert RJ et al (2011) Arabidopsis T-DNA insertional lines for CDC25 are hypersensitive to hydroxyurea but not to zeocin or salt stress. Ann Bot 107:1183–1192

    Article  PubMed  CAS  Google Scholar 

  148. Spadafora ND, Parfitt D, Li S et al (2012) Perturbation of cytokinin and ethylene-signalling pathways explain the strong rooting phenotype exhibited by Arabidopsis expressing the Schizosaccharomyces pombe mitotic inducer, cdc25. BMC Plant Biol 12:45

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  149. Price A, Orellana D, Salleh F et al (2008) A comparison of leaf and petal senescence in wallflower reveals common and distinct patterns of gene expression and physiology. Plant Physiol 147:1898–1912

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  150. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDC method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  151. Wagstaff C, Bramke I, Breeze E et al (2010) A specific group of genes respond to cold dehydration stress in cut Alstroemeria flowers whereas ambient dehydration stress accelerates developmental senescence expression patterns. J Exp Bot 61:2905–2921

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  152. Shinozaki K, Yamaguchi-Shinozaki K (2007) Gene networks involved in drought stress response and tolerance. J Exp Bot 58:221–227

    Article  PubMed  CAS  Google Scholar 

  153. Young DY, Udvardi M (2009) Translating Medicago truncatula genomics to crop legumes. Curr Opin Plant Biol 12:193–201

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biotechnology Research Center (BTRC), Tripoli, Libya

    Adel M. Elmaghrabi

  2. School of Biosciences, Cardiff University, Cardiff, UK

    Adel M. Elmaghrabi, Hilary J. Rogers & Dennis Francis

  3. Agroécologie, AgroSup Dijon, INRA, Univ. Bourgogne Franche-Comté, Dijon, France

    Sergio Ochatt

Authors
  1. Adel M. Elmaghrabi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hilary J. Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dennis Francis
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sergio Ochatt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sergio Ochatt .

Editor information

Editors and Affiliations

  1. CSIC-UPV, Institute for Plant Cell and Molecular Biology (IBMCP), Valencia, Spain

    Luis A. Cañas

  2. CSIC-UPV, Institute for Plant Cell and Molecular Biology (IBMCP), Valencia, Spain

    José Pío Beltrán

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Elmaghrabi, A.M., Rogers, H.J., Francis, D., Ochatt, S. (2018). Toward Unravelling the Genetic Determinism of the Acquisition of Salt and Osmotic Stress Tolerance Through In Vitro Selection in Medicago truncatula . In: Cañas, L., Beltrán, J. (eds) Functional Genomics in Medicago truncatula. Methods in Molecular Biology, vol 1822. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8633-0_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8633-0_19

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8632-3

  • Online ISBN: 978-1-4939-8633-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation