Responses of Arabica coffee (Coffea arabica L. var. Catuaí) cell suspensions to chemically induced mutagenesis and salinity stress under in vitro culture conditions
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.
KeywordsCoffea 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.
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.
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.
- 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. https://doi.org/10.1111/j.1752-4571.2012.00285.x CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1007/s11627-009-9254-2 CrossRefGoogle Scholar
- 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
- 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
- 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
- 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
- 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
- Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
- 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
- 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
- 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
- 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
- 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
- International Coffee Organization (2016) Total production by all exporting countries. http://www.ico.org/prices/po-production.pdf. Accessed 17 September 2016
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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) http://www.cenargen.embrapa.br/publica/trabalhos/ doc121.pdf Accessed 6 October 2016
- 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. https://doi.org/10.17660/ActaHortic.2011.897.41 CrossRefGoogle Scholar