Physiology and Molecular Biology of Plants

, Volume 24, Issue 5, pp 951–962 | Cite as

Development of an efficient in vitro mutagenesis protocol for genetic improvement of saffron (Crocus sativus L.)

  • Mahpara Kashtwari
  • Aijaz A. WaniEmail author
  • Manoj K. Dhar
  • Sabbi Jan
  • Azra N. Kamili
Research Article


Saffron (Crocus sativus L) is a triploid (2n = 3x = 24), sterile geophyte which can only be propagated by means of underground vegetative corms. Since corm multiplication does not induce genome variations, therefore, the entire saffron population is expected to have a similar genetic makeup. Keeping in view the economic importance of the plant and the factors responsible for its low yield, the present investigation has been undertaken to establish an in vitro ethyl methanesulfonate (EMS) mutagenesis protocol followed by characterization of the induced variability in the advanced generations. The present report is limited to standardization of in vitro mutagenesis protocol only. Among the mutagenic treatments tested, concentrations ranging from 0.1 to 0.5% EMS showed a varied survival of explants. Based on various growth parameters, the LD50 was calculated to be 0.3% EMS for 3 h. Among the two types of explants analyzed, the corm explant gave better results for in vitro survival and the growth parameters than callus explant. An average of 57.33 and 92.00 daughter cormlets in all EMS treatments as compared to 47.67 and 57.67 daughter cormlets in control, obtained from callus and corm explants respectively, were transferred to the field. The maximum, average daughter cormlet weight was obtained in control (3.01 g, corm explant) followed by 0.1% EMS (2.8 g, corm explant). In general, the growth parameters showed decreasing trend with an increase in EMS concentration in both the explants. The present study has been a significant achievement in the sense that the first mutagenesis protocol for C. sativus has been standardized.


Saffron EMS Mutagenesis Callus Shooting Cormlet 



We are highly thankful to Department of Biotechnology, Ministry of Science and Technology, Govt. of India for financial assistance in the project “Induction of variability for genetic improvement of Kashmir Saffron” vide File No. BT/PR5409/PBD/16/977/2012 dated 16.01.2013. Our sincere thanks is also due to in charge Tissue Culture Laboratory for providing necessary laboratory facility and to all those farmers who provided saffron material during our experimental work.


  1. Ahloowalia BS (1998) In-vitro techniques and mutagenesis for the improvement of vegetatively propagated plants. In: Jain SM, Brar DS, Ahloowalia BS (eds) Somaclonal variation and induced mutations in crop improvement. Current Plant Science and Biotechnology in Agriculture, vol 32. Springer, Dordrecht, pp 293–309CrossRefGoogle Scholar
  2. Ahuja A, Koul S, Ram G, Kaul BL (1994) Somatic embryogenesis and regeneration of plantlets in satiron. Indian J Exp Biol 32:135–140Google Scholar
  3. Bagheri AR (1989) Variation studies in saffron (Crocus sativus L) and its possible application in saffron breeding. IROST, MashadGoogle Scholar
  4. Basker D, Negbi M (1983) Uses of saffron. Econ Bot 37(2):228–236CrossRefGoogle Scholar
  5. Brandizzi F, Grilli Caiola M (1998) Flow cytometric analysis of nuclear DNA in Crocus sativus and allies (Iridacee). Plant Syst Evol 211:149–154CrossRefGoogle Scholar
  6. Brighton CA (1977) Cytology of Crocus sativus and its allies (Iridaceae). Plant Syst Evol 128(3–4):137–157CrossRefGoogle Scholar
  7. Broertjes C, Van Harten AM (2013) Applied mutation breeding for vegetatively propagated crops. vol 12. ElsevierGoogle Scholar
  8. Caiola MG, Canini A (2010) Looking for saffron’s (Crocus sativus L.) parents. Funct Plant Sci Biotechnol 4:1–14Google Scholar
  9. Castillo R, Fernandez JA, Gomez-Gomez L (2005) Implications of carotenoid biosynthetic genes in apocarotenoid formation during the stigma development of Crocus sativus and its closer relatives. Plant Physiol 139:674–689CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chichiriccò G (1984) Karyotype and meiotic behaviour of the triploid Crocus sativus L. Caryologia 37:233–239CrossRefGoogle Scholar
  11. Devi K, Sharma M, Singh M, Singh Ahuja P (2011) In vitro cormlet production and growth evaluation under greenhouse conditions in saffron (Crocus sativus L.)–a commercially important crop. Eng Life Sci 11(2):189–194CrossRefGoogle Scholar
  12. Dhar AK, Sapru R, Rekha K (1988) Studies on saffron in Kashmir 1: Variation in natural population and its cytological behaviour. Crop Improv 15:48–52Google Scholar
  13. Donini B (1982) Mutagenesis applied to improve fruit trees. Techniques, methods and evaluation of radiation-induced mutations. In: Induced mutations in vegetatively propagated plants IIGoogle Scholar
  14. Donini P, Sonnino A (1998) Induced mutation in plant breeding: current status and future outlook. In: Jain SM, Brar DS, Ahloowalia BS (eds) Somaclonal variation and induced mutations in crop improvement. Current Plant Science and Biotechnology in Agriculture, vol 32. Springer, Dordrecht, pp 255–291CrossRefGoogle Scholar
  15. Fernández JA (2004) Biology, biotechnology and biomedicine of saffron. Recent Res Dev Plant Sci 2:127–159Google Scholar
  16. George PS, Visvanath S, Ravishankar GA, Venkataraman LV (1992) Tissue culture of saffron (Crocus sativus L.): somatic embryogenesis and shoot regeneration. Food Biotechnol 6(3):217–223CrossRefGoogle Scholar
  17. Gismondi A, Fanali F, Labarga JM, Caiola MG, Canini A (2013) Crocus sativus L. genomics and different DNA barcode applications. Plant Syst Evol 299(10):1859–1863CrossRefGoogle Scholar
  18. Grilli Caiola M (2004) Saffron reproductive biology. Acta Hort 650:25–37CrossRefGoogle Scholar
  19. Grilli Caiola M (2005) Embryo origin and development in Crocus sativus L. (Iridacee). Plant Biosyst 139:335–343CrossRefGoogle Scholar
  20. Khan MA, Nagoo S, Naseer S, Nehvi FA, Zargar SM (2011) Induced mutation as a tool for improving corm multiplication in saffron (Crocus sativus L.). J Phytol 3:8–10Google Scholar
  21. Koch AC, Ramgareeb S, Rutherford RS, Snyman SJ, Watt MP (2012) An in vitro mutagenesis protocol for the production of sugarcane tolerant to the herbicide imazapyr. In Vitro Cell Dev Biol Plant 48:417–427CrossRefGoogle Scholar
  22. Mathew B (1977) Crocus sativus L. and its allies (Iridaceae). Plant Syst Evol 128:89–103CrossRefGoogle Scholar
  23. Milyaeva EL, Azizbekovas NS, Komarovam EN, Akhundova DD (1995) In vitro formation of regenerant corms of saffron crocus (Crocus sativus L.). Russ J Plant Physiol 42:112–1109Google Scholar
  24. Monika Y, Vinita R, Saharan RS, Sehrawat SK (2016) Influence of gamma radiations and EMS on morphological characteristics of gladiolus cv. PINK BEAUTY. AJH 11(1):114–118Google Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Nagaraju V, Bhowmik G, Parthasarathy VA (2002) Effect of paclobutrazol and sucrose on in vitro cormlet formation in gladiolus. Acta Bot Croat 61:27–33Google Scholar
  27. Nehvi FA (2003) Problems and prospects of saffron improvement in India. In: Proceedings of international seminar on industrial use of biotechnology. 27th September to 1st OctoberGoogle Scholar
  28. Nehvi FA, Khan MA, Lone AA, Maqhdoomi MI, Wani SA, Yousuf V, Yasmin S (2009) Effect of radiation and chemical mutagen on variability in saffron (Crocus sativus L.). In: III international symposium on saffron: forthcoming challenges in cultivation, research and economics, vol 850, pp 67–74Google Scholar
  29. Nehvi FA, Lone AA, Allai BA, Yasmin S (2010) Impact of climate change on saffron industry of Jammu and Kashmir. Crop Improv 37:203Google Scholar
  30. Novak FJ, Afza R, Duren MV, Omar MS (1990) Mutation induction by gamma irradiation of in vitro cultured shoot-tips of banana and plantain (Musa cvs). Trop Agric 67(1):21–28Google Scholar
  31. Parray JA, Kamili AN, Hamid R, Husaini AM (2012) In vitro cormlet production of saffron (Crocus sativus L. Kashmirianus) and their flowering response under greenhouse. GM Crops Food 3(4):289–295CrossRefPubMedGoogle Scholar
  32. Parray JA, Kamili AN, Hamid R, Reshi ZA, Qadri RA (2015) Antibacterial and antioxidant activity of methanol extracts of Crocus sativus L. cv. Kashmirianus. Front Life Sci 8(1):40–46CrossRefGoogle Scholar
  33. Pawar N, Nimbalkar M, Pai S, Kolar F, Dixit G (2010) RP-HPLC analysis of 6-gingerol and assessment of antioxidant activities in EMS treated ginger. J Phytol 2(3):13–23Google Scholar
  34. Perera D, Barnes DJ, Baldwin BS, Reichert NA (2015) Mutagenesis of in vitro cultures of Miscanthus × giganteus cultivar freedom and detecting polymorphisms of regenerated plants using ISSR markers. Ind Crop Prod 65:110–116CrossRefGoogle Scholar
  35. Plessner O, Ziv M, Negbi M (1990) In vitro corm production in the saffron crocus (Crocus sativus L.). Plant Cell Tissue Organ Cult 20(2):89–94CrossRefGoogle Scholar
  36. Quadri RR, Shah FA, Kamili AN, Shah AM (2008) In vitro response of corm and needle segments of saffron. J Himal Ecol Sustain Dev 3:94–104Google Scholar
  37. Quadri RR, Kamili AN, Husaini AM, Shah AM, Teixeira da Silva JA (2010) In vitro studies on cormogenesis and maximization of corm size in saffron. Funct Plant Sci Biotechnol 4:132–135Google Scholar
  38. Salwee Y, Nehvi FA (2014) Creation of genetic variability in saffron (Crocus sativus L.) using induced mutation. J Cell Tissue Res 14(2):4455–4461Google Scholar
  39. Sharma N, Kaur N, Gupta AK (1998) Effects of gibberellic acid and chlorocholine chloride on tuberisation and growth of potato (Solanum tuberosum L.). J Sci Food Agric 78:466–470CrossRefGoogle Scholar
  40. Silué S, Diarrassouba N, Fofana IJ, Muhovski Y, Toussaint A, Mergeai G, Jacquemin JM, Baudoin JP (2013) Description of Phaseolus vulgaris L. aborting embryos from ethyl methanesulfonate (EMS) mutagenized plants. Biotechnol Agron Soc Environ 17:563–571Google Scholar
  41. Simco I (1993) Effects of kinetin, paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light. Plant Growth Regul 12:23–27CrossRefGoogle Scholar
  42. Sung ZR (1976) Mutagenesis of cultured plant cells. Genetics 84(1):51–57PubMedPubMedCentralGoogle Scholar
  43. 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(12):1661CrossRefGoogle Scholar
  44. Winterhalter P, Straubinger M (2000) Saffron—renewed interest in an ancient spice. Food Rev Int 16(1):39–59CrossRefGoogle Scholar
  45. Zubor AA, Suranyi G, Gyori Z, Borbely G, Prokisch J (2004) Molecular biological approach of the systematic of Crocus sativus L. and its allies. Acta Hort 650:85–93CrossRefGoogle Scholar

Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2018

Authors and Affiliations

  1. 1.Cytogenetics and Reproductive Biology Laboratory, Department of BotanyUniversity of KashmirSrinagarIndia
  2. 2.Plant Genomics Laboratory, School of BiotechnologyUniversity of JammuJammuIndia
  3. 3.Department of Environmental Science, Centre of Research for DevelopmentUniversity of KashmirSrinagarIndia

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