In Vitro Cellular & Developmental Biology - Plant

, Volume 54, Issue 6, pp 576–589 | Cite as

Responses of Arabica coffee (Coffea arabica L. var. Catuaí) cell suspensions to chemically induced mutagenesis and salinity stress under in vitro culture conditions

  • Alejandro Bolívar-González
  • Marta Valdez-Melara
  • Andrés Gatica-AriasEmail author
Embryogenesis/Somatic Embryogenesis


Crop improvement of Coffea arabica L. (coffee) via mutagenesis could accelerate breeding programs; thus, the present study aimed to develop an in vitro protocol using the chemical mutagens sodium azide (NaN3) and ethyl methanesulfonate (EMS) on embryogenic cell suspensions of Arabica coffee variety Catuaí and, subsequently, to evaluate the responses of the resulting mutagenized tissues to salinity stress. Embryogenic suspension cultures were incubated with 0.0, 2.5, 5.0, or 10.0 mM NaN3 or 0.0, 185.2, 370.5, or 741.0 mM EMS. As the concentration of NaN3 or EMS increased, the survival of embryogenic suspension cultures decreased compared to controls. The median lethal dose (LD50) for NaN3 was 5 mM for 15 min and for EMS it was 185.2 mM for 120 min. Embryogenic suspension cultures treated with NaN3 or EMS were cultured on selective medium supplemented with 0, 50, 100, 150, 250, or 300 mM NaCl showed that 50 mM NaCl could be used as selection pressure. Plantlet growth and total amino acid content were affected by NaCl stress; some mutants had longer shoots and higher amino acid content than controls. Random amplified polymorphic DNA (RAPD) analysis was performed to determine whether the NaN3 or EMS treatments could induce genetic variability and resulted in identifiable polymorphic markers. A total of 18 10-mer primers were used to amplify genomic DNA of putative mutant and non-mutant arabica coffee embryogenic cultures and produced 50 scorable bands, of which 22% were polymorphic.


Coffea arabica L. embryogenic callus Chemical mutagenesis Ethyl methanesulfonate Sodium azide Salt stress 



The authors would like to thank Prof. Dr. Gerd Weber for his helpful comments on this manuscript.

Author contributions

A. B.-G. designed and performed the experiments, analyzed data, and wrote the paper; M. V.-M. conceived the project, discussed the results, and edited the paper; A. G.-A conceived the project, designed and coordinated the experiments, analyzed data, and wrote the paper.

Funding information

This study was financed by the University of Costa Rica and the Ministerio de Ciencia, Tecnología y Telecomunicaciones (MICITT) (project No. 111-B5-140).

Compliance with ethical standards

All authors read and approved the final manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Acanda Y, Martínez O, Prado MJ, González MV, Rey M (2014) EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine. Plant Cell Rep 33:471–481. CrossRefPubMedGoogle Scholar
  2. Aerts R, Berecha G, Gijbels P, Hundera K, van Glabeke S, Vandepitte K, Muys B, Roldán-Ruiz I, Honnay O (2012) Genetic variation and risks of introgression in the wild Coffea arabica gene pool in south-western Ethiopian montane rainforests. Evol Appl 6:243–252. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Albuquerque EVS, Cunha WG, Barbosa AEAD, Costa PM, Teixeira JB, Vianna GR, Cabral GB, Fernandez D, Grossi-de-Sa MF (2009) Transgenic coffee fruits from Coffea arabica genetically modified by bombardment. In Vitro Cell Dev Biol-Plant 45:532–539. CrossRefGoogle Scholar
  4. Appels R, Morris R, Gill B, May C (1998) Structural stability of chromosomes. In: Appels R, Morris R, Gill B, May C (eds) Chromosome biology, 1st edn. Springer International Publishing, New York, pp 92–95CrossRefGoogle Scholar
  5. Arena C, Turano M, Hay Mele B, Cataletto PR, Furia M, Pugliese M, De Micco V (2017) Anatomy, photochemical activity, and DNA polymorphism in leaves of dwarf tomato irradiated with X-rays. Biol Plant 61:305–314CrossRefGoogle Scholar
  6. Bartolomeo MP, Maisano F (2006) Validation of a reversed-phase HPLC method for quantitative amino acid analysis. J Biomol Tech 17:131–137PubMedPubMedCentralGoogle Scholar
  7. Behera M, Panigrahi J, Mishra RR, Rath SP (2012) Analysis of EMS induced in vitro mutants of Asteracantha longifolia (L.) Nees using RAPD markers. Indian J Biotechnol 11:39–47Google Scholar
  8. Bhumi S, Gautam S, Akshay R, Fenil P, Fougat RS (2013) Assessment of gamma radiation induced genetic variability in Jatropha curcas using RAPD and DAMD markers. Indian J Agr Sci 83:1381–1387Google Scholar
  9. Castro-Concha LA, Escobedo RM, Miranda-Ham ML (2006) Measurement of cell viability in in vitro cultures. In: Loyola-Vargas VM, Vázquez-Flota F (eds) Plant cell culture protocols, methods in molecular biology™, 2nd edn. Humana Press, New York, pp 71–76Google Scholar
  10. Chauhan N, Kumar D (2014) Effect of salinity stress on growth performance of Citronella java. Int J Geol Agric Environ Sci 2:11–14Google Scholar
  11. Chen S, Chai M, Jia Y, Gao Z, Zhang L, Gu M (2011) In vitro selection of salt tolerant variants following 60Co gamma irradiation of long-term callus cultures of Zoysia matrella [L.] Merr. Plant Cell Tissue Organ Cult 107:493–500CrossRefGoogle Scholar
  12. Chinnusamy V, Zhu JH, Zhu JK (2006) Salt stress signaling and mechanisms of plant salt tolerance. In: Setlow JK (ed) Genetic engineering, vol 27. Springer + Science Business Media Inc, New York, pp 141–177CrossRefGoogle Scholar
  13. Dhakshanamoorthy D, Selvaraj R, Chidambaram A (2010) Physical and chemical mutagenesis in Jatropha curcas L. to induce variability in seed germination, growth and yield traits. Rom J Biol Plant Biol 55:113–125Google Scholar
  14. Dhakshanamoorthy D, Selvaraj R, Chidambaram A (2013) Induced mutagenesis in Jatropha curcas L. using ethylmethanesulphonate (EMS) and assessment of DNA polymorphism through RAPD markers. J Crop Sci Biotechnol 16:201–207CrossRefGoogle Scholar
  15. Dhillon RS, Saharan RP, Jattan M, Rani T, Sheokand RN, Dalal V, Von Wuehlisch G (2014) Molecular characterization of induced mutagenesis through gamma radiation using RAPD markers in Jatropha curcas L. Afr J Biotechnol 13:806–813CrossRefGoogle Scholar
  16. dos Santos TB, Budzinski IGF, Marur CJ, Petkowicz CLO, Pereira LFP, Vieira LGE (2011) Expression of three galactinol synthase isoforms in Coffea arabica L. and accumulation of raffinose and stachyose in response to abiotic stresses. Plant Physiol Biochem 49:441–448CrossRefGoogle Scholar
  17. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  18. Duncan DB (1955) Multiple range and multiple F tests. Biometrics 11:1–42CrossRefGoogle Scholar
  19. Food and Agriculture Organization/International Atomic Energy Agency (2016) Plant breeding & genetic newsletter. Joint FAO/IAEA Programme for Nuclear Techniques in Food and Agriculture 36 http://www-nawebiaeaorg/nafa/pbg/public/pbg-nl-36pdf Accessed June 29, 2018
  20. Fernandez-Da Silva R, Menéndez-Yuffá A (2006) Viability in protoplasts and cell suspensions of Coffea arabica cv. Catimor. Electron J Biotechnol 9:593–597. CrossRefGoogle Scholar
  21. Figueirêdo VB, de Faria MA, da Silva EL (2006) Crescimento inicial do cafeeiro irrigado com água salina e salinização do solo. Rev Bras Eng Agríc Ambient 10:50–57CrossRefGoogle Scholar
  22. Galvão L, Silva E (2008) Randomly amplified polymorphic DNA (RAPD). In: Walker J, Rapley R (eds) Molecular biomethods handbook, 2nd edn. Humana Press, New York, pp 133–147CrossRefGoogle Scholar
  23. Ganesan M, Bhanumathi P, Jayabalan N (2005) Mutagenic effect of sodium azide on somatic embryo regeneration and root growth of cotton (Gossypium hirsutum L. CV. SVPR2). J Agr Technol 1:365–380Google Scholar
  24. Gatica-Arias AM, Arrieta G, Espinoza AM (2007) Comparison of three in vitro protocols for direct somatic embryogenesis and plant regeneration of Coffea arabica L. cvs. Caturra and Catuaí. Agron Costarric 31:85–94Google Scholar
  25. Gatica-Arias AM, Farag MA, Häntzschel KR, Matoušek J, Weber G (2012) The transcription factor AtMYB75/PAP1 regulates the expression of flavonoid biosynthesis genes in transgenic hop (Humulus lupulus L.). Brew Sci 65:103–111Google Scholar
  26. Ge H, Li Y, Fu H, Long G, Luo L, Li R, Deng Z (2015) Production of sweet orange somaclones tolerant to citrus canker disease by in vitro mutagenesis with EMS. Plant Cell Tissue Organ Cult 123:29–38. CrossRefGoogle Scholar
  27. Gruszka D, Szarejko I, Maluszynski M (2012) Sodium azide as a mutagen. In: Shu Q, Forster B, Nakagawa H (eds) Plant mutation breeding and biotechnology, 1st edn. CABI Publishing, Wallingford, pp 159–168CrossRefGoogle Scholar
  28. He S, Han Y, Wang Y, Zhai H, Liu Q (2009) In vitro selection and identification of sweetpotato (Ipomoea batatas (L.) Lam.) plants tolerant to NaCl. Plant Cell Tissue Organ Cult 96:69–74CrossRefGoogle Scholar
  29. Hofmann NE, Raja R, Nelson RL, Korban SS (2004) Mutagenesis of embryogenic cultures of soybean and detecting polymorphisms using RAPD markers. Biol Plant 48:173–177CrossRefGoogle Scholar
  30. Htwe NN, Maziah M, Ling HC, Qamaruz ZF, Mohd ZA (2011) Responses of some selected Malaysian rice genotypes to callus induction under in vitro salt stress. Afr J Biotechnol 10:350–362Google Scholar
  31. International Coffee Organization (2016) Total production by all exporting countries. Accessed 17 September 2016
  32. Jiménez VM (2005) Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis. Plant Growth Regul 47:91–110. CrossRefGoogle Scholar
  33. Khan IA, Dahot MU, Seema N, Yasmin S, Bibi S, Raza S, Khatri A (2009) Genetic variability in sugarcane plantlets developed through in vitro mutagenesis. Pak J Bot 41:153–166Google Scholar
  34. Koch A, Ramgareeb S, Rutherford R, Snyman S, Watt M (2012) An in vitro mutagenesis protocol for the production of sugarcane tolerant to the herbicide imazapyr. In Vitro Cell Dev Biol-Plant 48:417–427. CrossRefGoogle Scholar
  35. Kumar A, Kumar Naik G, Simmi PS, Giridhar P (2015) Salinity and drought response alleviate caffeine content of young leaves of Coffea canephora var. Robusta cv. S274. J Appl Biol Biotechnol 3:050–060Google Scholar
  36. Kumar K, Kumar M, Kim SR, Ryu H, Cho YG (2013) Insights into genomics of salt stress response in rice. Rice 6:27CrossRefGoogle Scholar
  37. Leitão J (2012) Chemical mutagenesis. In: Shu Q, Forster B, Nakagawa H (eds) Plant mutation breeding and biotechnology, 1st edn. CABI Publishing, Wallingford, pp 135–158CrossRefGoogle Scholar
  38. Loyola-Vargas VM (2016) The history of somatic embryogenesis. In: Loyola-Vargas VM, Ochoa-Alejo N (eds) Somatic embryogenesis: fundamental aspects and applications, 1st edn. Springer International Publishing, New York, pp 11–22CrossRefGoogle Scholar
  39. Loyola-Vargas VM, Avilez-Montalvo JR, Avilés-Montalvo RN, Márquez-López RE, Galaz-Ávalos RM, Mellado-Mojica E (2016) Somatic embryogenesis in Coffea spp. In: Loyola-Vargas VM, Ochoa-Alejo N (eds) Somatic embryogenesis: fundamental aspects and applications, 1st edn. Springer International Publishing, New York, pp 241–266CrossRefGoogle Scholar
  40. Luan YS, Zhang J, Gao XR, An LJ (2007) Mutation induced by ethylmethanesulphonate (EMS), in vitro screening for salt tolerance and plant regeneration of sweet potato (Ipomoea batatas L.). Plant Cell Tissue Organ Cult 88:77–81CrossRefGoogle Scholar
  41. Mansour SR, Abdel-Lateif K, Bogusz D, Franche C (2016) Influence of salt stress on inoculated Casuarina glauca seedlings. Symbiosis 70:129–138CrossRefGoogle Scholar
  42. Mba C, Afza R, Bado S, Jain SH (2010) Induced mutagenesis in plants using physical and chemical agents. In: Davey MR, Anthony P (eds) Plant cell culture: essential methods. Wiley, Chichester, pp 111–130CrossRefGoogle Scholar
  43. Mikula A, Niedzielski M, Rybczynski J (2006) The use of TTC reduction assay for assessment of Gentiana spp. cell suspension viability after cryopreservation. Acta Physiol Plant 28:315–324CrossRefGoogle Scholar
  44. Mishra M, Slater A (2012) Recent advances in the genetic transformation of coffee. Biotechnol Res Int 2012:1–17CrossRefGoogle Scholar
  45. Mishra P, Mishra V, Takabe T, Rai V, Kumar Singh N (2016) Elucidation of salt-tolerance metabolic pathways in contrasting rice genotypes and their segregating progenies. Plant Cell Rep 35:1273–1286CrossRefGoogle Scholar
  46. Morel G, Wetmore RH (1951) Fern callus tissue culture. Am J Bot 38:141–143CrossRefGoogle Scholar
  47. Mullainathan L, Sridevi A, Umavathi S, Sanjai Gandhi E (2014) Genetic variation in mutants of chilli (Capsicum annum) revealed by RAPD marker. Int Lett Nat Sci 6:1–8Google Scholar
  48. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681CrossRefGoogle Scholar
  49. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  50. Pádua M, Paiva L, Coutinho da Silva L, Livramento G, Alves E, Fonseca A (2014) Morphological characteristics and cell viability of coffee plants calli. Ciência Rural 44:660–665CrossRefGoogle Scholar
  51. Parida AK, Das AB (2005) Salt tolerance and salinity effects on plants: a review. Ecotoxicol Environ Saf 60:324–349CrossRefGoogle Scholar
  52. Parry M, Madgwick P, Bayon C, Tearall K, Hernandez-Lopez A, Baudo M, Rakszegi M, Hamada W, Al-Yassin A, Ouabbou H, Labhilili M, Phillips A (2009) Mutation discovery for crop improvement. J Exp Bot 60:2817–2825CrossRefGoogle Scholar
  53. Perrier X, Flor A, Bonnot F (2003) Methods of data analysis. In: Hamon P, Seguin M, Perrier X, Glaszmann JC (eds) Genetic diversity of cultivated tropical plants, 1st edn. Science Publishers, Montpellier, pp 43–76Google Scholar
  54. Quiroz-Figueroa FR, Fuentes CFJ, Rojas R, Loyola V (2002) Histological studies on the developmental stages and differentiation of two different somatic embryogenesis systems of Coffea arabica. Plant Cell Rep 20:1141–1149CrossRefGoogle Scholar
  55. Ribas AF, Luiz Filipe Protasio Pereira LFP, Vieira LGE (2011) Genetic transformation of coffee. Braz J Plant Physiol 18:83–94CrossRefGoogle Scholar
  56. Sandhu SS, Bastos CR, Azini LE, Neto AT, Colombo C (2002) RAPD analysis of herbicide-resistant Brasilian rice lines produced via mutagenesis. Genet Mol Res 1:359–370PubMedGoogle Scholar
  57. Senapati SK, Rout GR (2011) In vitro mutagenesis in Rosa hybrida using oryzalin as a mutagen and screening of mutants by randomly amplified polymorphic DNA (RAPD) marker. Afr J Biotechnol 10:5705–5712Google Scholar
  58. Serrat X, Esteban R, Guibourt N, Moysset L, Nogués S, Lalanne E (2014) EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations. Plant Methods 10:5. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Sheikh D, Alborzian A, Moradnejad M (2014) Mutagenesis in olive (Olea europaea L.) calli caused by sodium azide and detection of mutants using ISSR and RAPD markers. J Hortic Sci Biotechnol 89:153–158CrossRefGoogle Scholar
  60. Silva MC, Várzea V, Guerra-Guimarães L, Azinheira HG, Fernandez D, Petitot AS, Bertrand B, Lashermes P, Nicole M (2006) Coffee resistance to the main diseases: leaf rust and coffee berry disease. Braz J Plant Physiol 18:119–147CrossRefGoogle Scholar
  61. Suprasanna P, Mirajkar S, Bhagwat S (2015) Induced mutations and crop improvement. In: Bahadur B, Rajam M, Sahijram L, Krishnamurthy K (eds) Plant biology and biotechnology. Vol I: Plant Diversity, Organization, Function and Improvement, 1st edn. Springer International Publishing, New York, pp 593–617CrossRefGoogle Scholar
  62. Talebi AB, Talebi AB, Shahrokhifar B (2012) Ethyl methane sulphonate (EMS) induced mutagenesis in Malaysian rice (cv. MR219) for lethal dose determination. Am J Plant Sci 3:1661–1665CrossRefGoogle Scholar
  63. Teixeira JB, Junqueira CS, Pereira JPC, Mello S, Silva PD, Mundim DA (2004) Multiplicação clonal de café (Coffea arabica L.) via embryogenesis somática. Brasília: Embrapa Recursos Genéticos e Biotecnologia. (Embrapa Recursos Genéticos e Biotecnologia. Documentos, 121) doc121.pdf Accessed 6 October 2016
  64. Towill LE, Mazur P (1975) Studies on the reduction of 2,3,5-triphenyl tetrazolium chloride as a viability assay for plant tissue cultures. Can J Bot 53:1097–1102CrossRefGoogle Scholar
  65. van Boxtel J, Berthouly M (1996) High frequency somatic embryogenesis from coffee leaves. Plant Cell Tissue Organ Cult 44:7–17CrossRefGoogle Scholar
  66. Wannajindaporn A, Poolsawat O, Chaowiset W, Tantasawat P (2014) Evaluation of genetic variability in in vitro sodium azide-induced Dendrobium ‘Earsakul’ mutants. Gen Mol Res 13:5333–5342CrossRefGoogle Scholar
  67. Xu C, Xiao J, He J, Hu G, Chen H (2011) The effect of ethyl methane sulphonate (EMS) and sodium azide (NaN3) on plant regeneration capacity of an embryogenic cell suspension of ‘Yueyoukang 1’ (Musa, aaa), a banana cultivar resistant to fusarium wilt. Acta Hortic 897:301–302. CrossRefGoogle Scholar
  68. Yuang-Ling C, Hui L, Xing M, Su L, Yong X, Zhen L, Le C, Yao L (2013) An efficient rice mutagenesis system based on suspension-cultured cells. J Integr Plant Biol 55:122–130CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  • Alejandro Bolívar-González
    • 1
  • Marta Valdez-Melara
    • 1
  • Andrés Gatica-Arias
    • 1
    Email author
  1. 1.Laboratorio de Biotecnología de Plantas, Escuela de BiologíaUniversidad de Costa RicaSan PedroCosta Rica

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