Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 117, Issue 3, pp 323–334 | Cite as

Plant regeneration from protoplasts in Indian local Coriandrum sativum L.: scanning electron microscopy and histological evidences for somatic embryogenesis

  • A. MujibEmail author
  • Dipti Tonk
  • Muzamil Ali
Original Paper


Coriandrum sativum L. is an annual herb belonging to the family Umbelliferae. It is used as a spice plant in Indian subcontinent and it has several medicinal applications as well. In this present article, an efficient plant regeneration protocol from protoplasts via somatic embryogenesis was established and is reported. This is the first ever protoplast isolation study in Indian local coriander in which plant regeneration was achieved. Hypocotyl-derived embryogenic callus was used as a source of protoplast. The embryogenic callus suspension was prepared by transferring tissues onto rotary-agitated liquid Murashige and Skoog, added with 1.0 mg l−1 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l−1 KIN (6-furfurylaminopurine). The suspension was digested with enzymatic solutions and a combination of cellulase (2.0 %), pectinase (1.0 %), macerozyme (0.02 %) and driselase (0.50 %) induced maximum yield of protoplasts (34.25 × 105). In 1.0 mg l−1 2,4-D + 1.0 mg l−1 KIN containing medium, protoplasts divided well and formed maximum number of microcolonies (14.30/test tube). The protoplast callus (PC) biomass grew well in solid medium. The protoplast embryogenic callus was rich in protein, proline and sugar compared to non-embryogenic PC. The protoplast originated callus later differentiated into somatic embryos. The somatic embryo morphology, scanning electron microscopy and histology of embryo origin and development were investigated and discussed in details in this present communication. In 1.0 mg l−1 2,4-D + 0.5 mg l−1 BA (6-Benzyladenine), maximum number of embryos were formed on microcallus (26.6/callus mass). The embryo matured and germinated into plantlets at a low to moderate rate, highest (31.3 %) embryo germination was observed in 1.0 mg l−1 BA + 0.5 mg l−1 α-Naphthalene acetic acid added medium. The entire process of regeneration took about 4–5 months’ time for recovering plantlets from protoplasts.


Coriandrum sativum Protoplasts Enzymatic digestion Micro-colony Somatic embryos Scanning electron microscopy Histology 



2,4-Dichlorophenoxyacetic acid






Murashige and Skoog


α-Naphthalene acetic acid


Plant growth regulator


Scanning electron microscopy



The authors are thankful to University Grant Commission (UGC) and Department of Botany, Hamdard University (Jamia Hamdard) for receiving facilities of various kinds. The first author also acknowledges the help, rendered by M.Sc and Ph.D students.


  1. Alhubaishi AA, Walkey DGA, Webb MJW, Bolland CJ, Cook AA (1987) A survey of horticultural plant virus diseases in the Yemen Arab Republic. FAO Plant Prot Bull 35:135–143Google Scholar
  2. Assani A, Haicour R, Wenzel G, Foroughi-Wehr B, Bakry F, Cote FX, Ducreux G, Ambroise A, Grapin A (2002) Influence of donor material and genotype on protoplast regeneration in banana and plantain cultivars (Musa spp.). Plant Sci 162:355–362CrossRefGoogle Scholar
  3. Aziz ZA, Davey MR, Lowe KC, Power JB (2006) Isolation and culture of protoplasts from the medicinal plant Centella asiatica. Rev Bras Pl Med 8:105–109Google Scholar
  4. Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207CrossRefGoogle Scholar
  5. Bhatnagar SS (1950) Coriandrum Linn. (Umbelliferae). In: Bhatnagar SS (ed) The wealth of India. A dictionary of indian raw materials and industrial products, raw materials, vol 2. Council of Scientific and Industrial Research, New Delhi, pp 347–350Google Scholar
  6. Borgato L, Pisani F, Furini A (2007) Plant regeneration from leaf protoplasts of Solanum virginianum L. (Solanaceae). Plant Cell Tiss Org Cult 88:247–252CrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for measuring quantities of protein utilizing the principle of protein dye binding. Ann Biochem 72:248–253CrossRefGoogle Scholar
  8. Burns JA, Wetzstein HY (1995) Development and germination of pecan somatic embryos derived from liquid culture. In Vitro Cell Dev Biol-Plant 31:72–78CrossRefGoogle Scholar
  9. Cabasson C, Alvard D, Dambier D, Ollitrault P, Teisson C (1997) Improvement of Citrus somatic embryo development by temporary immersion. Plant Cell Tiss Organ Cult 50:33–37CrossRefGoogle Scholar
  10. Christina ENL, Arunika H (2010) Isolation of leaf protoplasts from the submerged aquatic monocot Aponogeton madagascariensis. Am J Pl Sci Biotech 4:6–11Google Scholar
  11. Cislova LS (1988) Useful types of coriander (in Russian). Sel Semenovod (Moskva), 33Google Scholar
  12. Cocking EC (1960) Enzymatic degradation of cell wall for protoplast formation. Nature 187:927–929CrossRefGoogle Scholar
  13. Das A, Mallick R (1989) Variation in 4C DNA content and chromosome characteristics in different varieties of Coriandrum sativum L. Cytologia 54:609–616CrossRefGoogle Scholar
  14. Davey MR, Anthony P, Power JB, Lowe JB (2005) Plant protoplast technology: current status. Acta Physiol Plant 27:117–129CrossRefGoogle Scholar
  15. Dey PM (1990) Methods in plant biochemistry, carbohydrates, vol 2. Academic Press, LondonGoogle Scholar
  16. Diederichsen A (1996) Coriander (Coriandrum sativum L.). Promoting the conservation and use of underutilized and neglected crops, vol 3. Institute of Plant Genetics and Crop Plant Research, Gatersleben/International Plant Genetic Resources Institute, RomeGoogle Scholar
  17. Dietert MF, Barron SA, Yoder OC (1982) Effects of genotype on in vitro culture in the genus Brassica. Plant Sci Lett 26:233–240CrossRefGoogle Scholar
  18. Dovzhenko A, Dal Bosco C, Meurer J, Koop HU (2003) Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana. Protoplasma 222:107–111PubMedCrossRefGoogle Scholar
  19. Dipak KP, Swati ND, Hardik PG, Rangitsinh VD, Ramachandra AV (2012) Cardio protective effects of C. sativum on isoproterenol induced myocardial necrosis in rats. Food Chem Toxicol 50:3120–3125Google Scholar
  20. Duarte A, Ferreira S, Silva F, Domingue FC (2012) Synergistic activity of coriander oil and conventional antibiotics against Acinetobacter baumannii. Phytomedicine 19:236–238Google Scholar
  21. Duquenne B, Eeckhaut T, Werbrouck S, Van Huylenbroeck J (2007) Effect of enzyme concentrations on protoplast isolation and protoplast culture of Spathiphyllum and Anthurium. Plant Cell Tiss Organ Cult 91:165–173CrossRefGoogle Scholar
  22. Ewa G, Marek S, Rafal B (2012) An improved protocol for plant regeneration from leaf and hypocotyl-derived protoplasts of carrot. Plant Cell Tiss Organ Cult 109:101–109CrossRefGoogle Scholar
  23. Fayez KA, Hassanein AM (2000) Chlorophyll synthesis retardation and ultra structural alterations to Solanum tuberosum chloroplasts in Solanum nigrum cells. Photosynthetica 38:37–44CrossRefGoogle Scholar
  24. Fiuk A, Rajkiewicz M, Rybczynski JJ (2003) In vitro culture of Gentiana kurroo (Royle). Biotech 3:267–274Google Scholar
  25. Fock I, Collonnier C, Purwito A, Luisetti J, Souvannavong V, Vedel F, Servaes A, Ambroise A, Kodja H, Ducreux G, Sihachakr D (2000) Resistance to bacterial wilt in somatic hybrids between Solanum tuberosum and Solanum phureja. Plant Sci 160:165–176PubMedCrossRefGoogle Scholar
  26. Gaj MD (2004) Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Heynh. J Plant Growth Regul 43:27–47CrossRefGoogle Scholar
  27. Girenko MM (1982) Initial material and basic trends in breeding of some uncommon species of vegetables. Bull VIR im Vavilova 120:33–37 (in Russian, English abstract)Google Scholar
  28. Guo JM, Liu QC, Zhai H, Wang YP (2006) Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam. Plant Cell Tiss Organ Cult 87:321–327CrossRefGoogle Scholar
  29. Hall RD, Riksen-Bruinsma T, Weyens GJ, Rosquin IJ, Denys PN, Evans IJ, Lathouwers JE, Lefcbvre MP, Dunwell JM, van Tunen A, Krens FA (1996) A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells. Nat Biotechnol 14:1133–1138PubMedCrossRefGoogle Scholar
  30. Ilyas M (1980) Spices in India. Econ Bot 34:236–259CrossRefGoogle Scholar
  31. Ivanova KV, Stoletova EA (1990) The history of culture and intraspecific taxonomy of Coriandrum sativum L. Sb nau n tr prikl bot, gen i sel 133:26–40 (in Russian, English abstract)Google Scholar
  32. Jeffery LD, Karl DH, Susanne L, Klaus H, Dierk S (1987) Parsley protoplasts retain differential responsiveness to u.v. light and fungal elicitor. EMBO J 6:2551–2556Google Scholar
  33. Jiménez VM, Thomas C (2006) Participation of plant hormones in determination and progression of somatic embryogenesis. In: Mujib A, Samaj J (eds) Somatic embryogenesis. Springer-Verlag, Heidelberg, pp 103–118CrossRefGoogle Scholar
  34. Junaid A, Mujib A, Bhat MA, Sharma MP, Šamaj J (2007) Somatic embryogenesis and plant regeneration in Catharanthus roseus. Biol Plant 51:641–646CrossRefGoogle Scholar
  35. Kantharajah AS, Golegaonkar PJ (2004) Somatic embryogenesis in egg plant. Sci Hortic 99:107–117CrossRefGoogle Scholar
  36. Karami O, Deljao A, Pour AM (2007) Repetitive somatic embryogenesis in carnation in picloram supplemented media. Plant Growth Regul 51:33–39CrossRefGoogle Scholar
  37. Karamian R, Ebrahimzadeh H (2001) Plantlet regeneration from protoplast-derived embryogenic calli of Crocus cancellatus. Plant Cell Tiss Organ Cult 65:115–121CrossRefGoogle Scholar
  38. Kim SW, Seung CO, Dong SI, Liu JR (2003) Plant regeneration of rose (Rosa hybridia) from embryogenic cell-derived protoplasts. Plant Cell Tiss Organ Cult 73:15–19CrossRefGoogle Scholar
  39. Kohlenbach HW, Wenzel G, Hoffmann F (1982) Regeneration of Brassica napus plantlets in cultures from isolated protoplasts of haploid stem embryos as compared with leaf protoplasts. Zeitschrift Pflanzen Physiol 105:131–142CrossRefGoogle Scholar
  40. Li JS, Deng MX, Mao ZHI, Hong YU (2005) En-hanced anthocyanins synthesis in foliage plant Ca-ladium bicolor. Plant Cell Rep 23:716–720PubMedCrossRefGoogle Scholar
  41. Liu F, Ryschka U, Marthe F, Klocke E, Schumann G, Zhao H (2007) Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis. Protoplasma 231:89–97PubMedCrossRefGoogle Scholar
  42. Lörincz K, Tyihák E (1965) Untersuchungen über die Terpenkomponenten im Verlaufe der Ontogenie des Korianders (Coriandrum sativum L.). Herba Hung 4:191–208 (in Russian, German abstract, Hungarian abstract)Google Scholar
  43. Ma R, Ritala A, Caldentey KMO, Rischer H (2006) Development of in vitro techniques for the important medicinal plant Veratrum californicum. Planta Med 72:1142–1148PubMedCrossRefGoogle Scholar
  44. Mataiyoh JC, Maiyo ZC, Ngure RM, Chepkorir R (2009) Chemical composition and antibacterial activity of the essential oil of C.sativum. Food Chem 113:526–529Google Scholar
  45. Mathias ME (1994) Magic, myth and medicine. Econ Bot 48:3–7CrossRefGoogle Scholar
  46. Mehpara M, Mujib A, Dipti T, Abdin MZ (2012) Protoplast isolation, culture and plant regeneration in Catharanthus roseus (L.) G. Don via somatic embryogenesis. Curr Biotechnol 1(3):217–226CrossRefGoogle Scholar
  47. Mizuhiro M, Kenichi Y, Ito K, Kadowaki S, Ohashi H, Mii M (2001) Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Pimula obconica. Plant Sci 160:1221–1228PubMedCrossRefGoogle Scholar
  48. Mujib A, Bandyopadhyay S, Jana BK, Ghosh PD (1996) Direct somatic embryogenesis and in vitro plant regeneration in Hippeastrum hybridum. Plant Tissue Cult 8:19–25Google Scholar
  49. Mujib A, Banerjee S, Maqsood M, Ghosh PD (2013) Somatic embryogenesis of some member ornamental genera of Amaryllidaceae and allied families: the similarities and differences. The Open Hort J 6:9–18CrossRefGoogle Scholar
  50. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  51. Nieves N, Segura-nieto M, Blanco MA, Sanchez M, Gonzales A, Gonzales JL, Castillo R (2003) Biochemical characterization of embryogenic and nonembryogenic calluses of sugarcane. In Vitro Cell Dev Biol-Plant 39:343–345CrossRefGoogle Scholar
  52. Oropeza M, Marcano AK, De Garcia E (2001) Proteins related in embryogenic potentials in callus and cell suspensions of sugarcane (Sacharrum sp.). In Vitro Cell Dev Biol-Plant 37:211–216CrossRefGoogle Scholar
  53. Pan ZG, Liu CZ, Murch SJ, El-Demerdash M, Saxena PK (2003) Plant regeneration from mesophyll protoplasts of the Egyptian medicinal plants Artmesia judaica L. and Echinops spinosissimus Turra. Plant Sci 165:681–687CrossRefGoogle Scholar
  54. Paruthi IJ, Jain RK, Gupta DC (1987) A note on reaction of some spices to root knot nematode (Meloidogyne javanica). Haryana J Hortic Sci 16:154–155Google Scholar
  55. Pongchawee K, Na-Nakorn U, Laamseejan S, Poompuang S, Phansiri S (2007) Protoplast isolation and culture of aquatic plant Cryptocoryne wendtii De Wit. Kasetsart J NatSci 41:300–310Google Scholar
  56. Prakash V (1990) Leafy spices. CRC Press Inc, Boca RatonGoogle Scholar
  57. Ramanayake SMSD, Wanniarachchi WAVR, Tennakoon TMA (2001) Axillary shoot proliferation and in vitro flowering in an adult giant bamboo, Dendrocalamus giganteus Wall. Ex Munro. In Vitro Cell Dev Biol-Plant 37:667–671CrossRefGoogle Scholar
  58. Shrestha BR, Tokuhara K, Mii M (2007) Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis. Plant Cell Rep 26:719–725PubMedCrossRefGoogle Scholar
  59. Silva F, Ferreira S, Duarte A, Mendonca DI, Domingue FC (2011) Antifungal activity of C. sativum essential oil, its mode of action against candida species and potential synergism with amphotericin B. Phytomedicine 19:42–47Google Scholar
  60. Slaby K, Havel L (1999) Conversion of somatic embryos of Picea abies (L.). Karst. Biologia 54:7–15Google Scholar
  61. Soumendra NT, Subham D, Gungan G, Moumita A, Aniruddha M (2010) Therapeutic efficacies of C. sativum aqueous extract against metronidazole induced genotoxicity in Channa punctatus peripheral erythrocytes. Food Chem Toxicol 48:3458–3461Google Scholar
  62. Sreelatha S, Padma PR, Umadevi M (2009) Protective effects of C. Sativum extract on carbon tetrarchloride induced hepatotoxicity in rats. Food Chem Toxicol 47:702–708Google Scholar
  63. Te-Chato S, Hilae A, Moosikapala L (2005) Microcolony formation from embryogenic callus-derived protoplasts of oil palm. Songklanakarin J Sci Technol 27:685–691Google Scholar
  64. Thomas TD (2009) Isolation, callus formation and plantlet regeneration from mesophyll protoplasts of Tylophora indica (Burm. f.) Merrill: an important medicinal plant. In Vitro Cell Dev Biol-Plant 45:591–598CrossRefGoogle Scholar
  65. Toben HM (1995) Die durch Pseudomonas syringae pv. coriandricola hervorgerufene Doldenwelke an Koriander, Charakterisierung des Erregers und Strategien zur Bekämpfung. Cuvillier Verlag, GöttingenGoogle Scholar
  66. Vavilov NI (1992) The phyto-geographical basis for plant breeding—studies of the original material used for plant breeding. In: Dorofeev VF (ed) Origin and geography of cultivated plants. Cambridge University Press, Cambridge, pp 316–366Google Scholar
  67. Yan CQ, Qian KX, Yan QS, Zhang XQ, Xue GP, Huangfu WG, Wu YF, Zhao YZ, Xue ZY, Huang J, Xu GZ, Wu P (2004) Use of asymmetric somatic hybridization for transfer of the bacterial blight resistance trait from Oryza meyeriana L. to O. sativa L. ssp. japonica. Plant Cell Rep 22:569–575PubMedCrossRefGoogle Scholar
  68. Yang X, Guo X, Zhang X, Nie Y, Jin S (2007) Plant regeneration from Gossypium davidsonii protoplasts via somatic embryogenesis. Biol Plant 51:533–537CrossRefGoogle Scholar
  69. Zohra F, Mujib A, Samar F, Arshi A, Umar S (2009) Callus induction, biomass growth, and plant regeneration in Digitalis lanata Ehrh.: influence of plant growth regulators and carbohydrates. Turk J Bot 33:393–405Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Cellular Differentiation and Molecular Genetics Section, Department of BotanyHamdard UniversityNew DelhiIndia

Personalised recommendations