Plant Biotechnology Reports

, Volume 7, Issue 3, pp 277–286 | Cite as

Morpho-histological and ultrastructural study on direct somatic embryogenesis of Capsicum chinense Jacq. in liquid medium

  • Susana A. Avilés-Viñas
  • Carlos A. Lecona-Guzmán
  • Adriana Canto-Flick
  • Stephanie López-Erosa
  • Nancy Santana-BuzzyEmail author
Original Article


Somatic embryo-like structures were produced from the hypocotyls of aseptic plants of Capsicum chinense. Different concentrations of 2,4-dichlorophenoxyacetic acid (0, 4.5, 9.05 μM), several exposure times of the explant to this auxin (15, 30, 45, 60 days) and the development of somatic embryos cultured in a solid and/or liquid medium were evaluated. As a result, a novel system of regeneration via direct somatic embryogenesis in liquid medium was established, with an efficiency of 1.77 × 104 somatic embryos per liter of medium. Critical stages of embryogenesis, including cellular acquisition of morphogenetic competence, suspensor formation, and development and maturation of somatic embryos, were identified by histological analysis and scanning electron microscopy. Our results show a promising new outlook on the in vitro regeneration of this species. Contrary to what has been reported to date for the Capsicum genus, it is a species of plants with higher embryogenic potential in vitro.


Habanero pepper Somatic embryos Histology Scanning electron microscopy 



2,4-Dichlorophenoxyacetic acid


Murashige and Skoog medium


Somatic embryogenesis


Somatic embryos


Scanning electron microscope



We wish to thank CONACYT and FOMIX for providing financial support.


  1. Agrawal S, Chandra N, Kothari SL (1989) Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. Mathania). Plant Cell Tissue Organ Cult 116:47–55CrossRefGoogle Scholar
  2. Auboiron E, Carron MP, Michaux-Ferriere N (1990) Influence of atmospheric gases, particularly ethylene, on somatic embryogenesis of Hevea brasiliensis. Plant Cell Tissue Organ Cult 21:31–37CrossRefGoogle Scholar
  3. Barwale UB, Kerns HR, Widholm JM (1986) Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis. Planta 167:473–481CrossRefGoogle Scholar
  4. Berlin GP, Miksche JP (1976) Botanical microtechnique and cytochemistry, 3rd edn. Iowa State University Press, AmesGoogle Scholar
  5. Binzel ML, Sankhla N, Joshi S, Sankhla D (1996) Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.). Plant Cell Rep 15:536–540CrossRefGoogle Scholar
  6. Buyukalaca S, Mavituna F (1996) Somatic Embryogenesis and plant regeneration of pepper in liquid media. Plant Cell Tissue Org Cult 46:227–235CrossRefGoogle Scholar
  7. Decout E, Dubois T, Guedira M, Dubois J, Audran JC, Vasseur J (1994) Role of temperature as a triggering signal for organogenesis or somatic embryogenesis in wounded leaves of chicory cultured in vitro. J Expt Bot 45:1859–1865CrossRefGoogle Scholar
  8. Dos Santos AVP, Cutter EG, Davey MR (1983) Origin and development of somatic embryos in Medicago sativa L. (alfalfa). Protoplasma 117:107–115CrossRefGoogle Scholar
  9. Feher A, Pasternak TP, Dudits D (2003) Transition of somatic cells to an embryogenic state. Plant Cell Tissue Organ Cult 74(3):201–228CrossRefGoogle Scholar
  10. Finer JJ, McMullen MD (1991) Transformation of soybean via particle bombardment of embryogenic suspension culture tissue. In Vitro Cell Dev Biol 27:175–182Google Scholar
  11. Fischer-Iglesias C, Sundberg B, Neuhaus G, Jones AM (2001) Auxin distribution and transport during embryonic pattern formation in wheat. Plant J 26:115–129PubMedCrossRefGoogle Scholar
  12. Fransz PF, Schel JHN (1991) An ultrastructural study on the early development of Zea mays somatic embryos. Can J Bot 69(4):858–865CrossRefGoogle Scholar
  13. Gaj M (2004) Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to A. thaliana (L.) Heynh. Plant Growth Regul 43:27–47CrossRefGoogle Scholar
  14. Gill R, Saxena PK (1992) Direct somatic embryogenesis and regeneration of plants from seedling explants of peanut (Arachis hypogaea): promotive role of thidiazuron. Can J Bot 70(6):1186–1192CrossRefGoogle Scholar
  15. Guzzo F, Baldan B, Mariani P, Lo Schiavo F, Terzi M (1994) Studies on the origin of totipotent cells in explants of Daucus Carota L. J Expt Bot 45:1427–1432CrossRefGoogle Scholar
  16. Halperin W, Jensen WA (1967) Ultrastructural changes during growth and embryogenesis in carrot cell cultures. J Ultrastruct Res 18:428–443PubMedCrossRefGoogle Scholar
  17. Harini I, Sita L (1993) Direct somatic embryogenesis and plant regeneration from immature embryos of chilli (C. annuum L). Plant Sci 89:107–112CrossRefGoogle Scholar
  18. Heidmann I, de Lange B, Lambalk J, Angenent GC, Boutilier K (2011) Efficient sweet pepper transformation mediated by the BABY BOOM transcription factor. Plant Cell Rep 30:1107–1115PubMedCrossRefGoogle Scholar
  19. Husain S, Jain A, Kothari SL (1999) Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annuum L. Plant Cell Rep 19:64–68CrossRefGoogle Scholar
  20. Jimenez VM (2005) Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis. Plant Growth Reg 47:91–110Google Scholar
  21. Jo J, Choi E, Choi D, Lee K (1996) Somatic embryogenesis and plant regeneration from immature zygotic embryo culture in pepper (C. annuum L.). J Plant Biol 39:127–135Google Scholar
  22. Kaparakis G, Alderson PG (2008) Role for cytokinins in somatic embryogenesis of pepper (Capsicum annuum L.)? J Plant Growth Regul. 27:110–114Google Scholar
  23. Khalil SM, Cheah KT, Perez EA, Gaskill DA, Hu JS (2002) Regeneration of banana (Musa spp. AAB cv. Dwarf Brazilian) via secondary somatic embryogenesis. Plant Cell Rep 20:1128–1134Google Scholar
  24. Kintzios S, Drossopolous J, Lymperopoulos C (2001) Effect of vitamins and inorganic micronutrients on callus growth and somatic embryogenesis from leaves of chilli pepper. Plant Cell Tissue Org Cult 67:55–62CrossRefGoogle Scholar
  25. Kiss E, Heszky LE, Gyulai G, Horváth HS, Csillag A (1991) Neomorph and leaf differentiation as alternative morphogenetic pathways in Soybean tissue culture. Acta Biol Hung 42:313–321PubMedGoogle Scholar
  26. Kothari SL, Kachhwaha AJS, Ochoa-Alejo N (2010) Chilli peppers—a review on tissue culture and transgenesis. Biotechnol Adv 28(1):35–48PubMedCrossRefGoogle Scholar
  27. Liu W, Parrott W, Hildebrand D, Collins G, Williams E (1990) Agrobacterium-induced gall formation in bell pepper (Capsicum annuum L.) and formation of shoot-like structures expressing introduced genes. Plant Cell Rep 9:360–364Google Scholar
  28. López-Puc G, Canto-Flick A, Barredo-Pool F, Zapata-Castillo P, Montalvo-Peniche M, Barahona-Pérez F, Santana-Buzzy N (2006) Direct somatic embryogenesis: a highly efficient protocol for in vitro regeneration of Habanero pepper (Capsicum chinense Jacq.). HortScience 41(6):1–7Google Scholar
  29. Maximova SN, Alemanno L, Young A, Ferriere N, Traore A, Guiltinan MJ (2002) Efficiency, genotypic variability, and cellular origin of primary and secondary Somatic embryogenesis of theobroma cacao l. In Vitro Cell Dev Biol 38:252–259Google Scholar
  30. Michalczuk L, Ribnicky D, Cooke T, Cohen D (1992) Regulation of indole-3-acetic acid biosynthetic pathways in carrot cell cultures. Plant Physiol 100:1346–1353PubMedCrossRefGoogle Scholar
  31. Murashige T, Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plantarum 15(3):437–497CrossRefGoogle Scholar
  32. Nissen P, Minocha SC (1993) Inhibition by 2,4-D of somatic embryogenesis in carrot as explored by its reversal by difluoromethylornithine. Physiol Plant 89:673–680CrossRefGoogle Scholar
  33. Ochoa-Alejo N, Ireta-Moreno L (1990) Cultivar differences in shoot-forming capacity of hypocotyl tissues of chilli pepper (Capsicum annuum L.) cultured in vitro. Sci Hortic-Amsterdam 42:21–28CrossRefGoogle Scholar
  34. Ochoa-Alejo N, Ramírez-Malagón R (2001) In vitro chili pepper biotechnology. In Vitro Cell Dev Biol Plant 37(6):701–729CrossRefGoogle Scholar
  35. Pasternak T, Prinsen E, Aydin F, Miskolczi P, Potters G, Asard H, Van Onckelen H, Dudits D, Fehér A (2002) The role of auxins, pH and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa. Plant Physiol 129:1807–1819PubMedCrossRefGoogle Scholar
  36. Quiroz-Figueroa FR, Rojas-Herrera R, Galaz-Avalos RM, Loyola-Vargas VM (2006) Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant Cell Tissue Org Cult 86(3):285–301CrossRefGoogle Scholar
  37. Ramírez-Malagón R, Ochoa-Alejo N (1996) An improved and reliable chilli pepper (Capsicum annuum L.) plant regeneration method. Plant Cell Rep 16:226–231CrossRefGoogle Scholar
  38. Santana-Buzzy N, Canto-Flick A, Iglesias-Andreu LG, Montalvo-Peniche MC, López-Puc G, Barahona-Pérez F (2006) Improvement of in vitro culturing of Habanero pepper by inhibition of ethylene effects. HortScience 41(2):405–409Google Scholar
  39. Santana-Buzzy N, Lopez-Puc G, Canto-Flick A, Barredo-Pool F, Balam-Uc E, Aviles-Viñas S, Solís-Marroquín D, Lecona-Guzman C, Bello-Bello J, Gomez-Uc E, Mijangos-Cortes JO (2009) Ontogenesis of the somatic embryogenesis of Habanero pepper (Capsicum chinense Jacq.). HortScience 44(1):113–118Google Scholar
  40. Sato S, Newell C, Kolacz K, Tredo L, Finer J, Hinchee M (1993) Stable transformation via particle bombardment in two different soybean regeneration systems. Plant Cell Rep 12:408–413Google Scholar
  41. Steinitz B, Küsek M, Tabib Y, Paran I, Zelcer A (2003) Pepper (C. annuum L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot. In Vitro Cell Dev Biol Plant 39:296–303CrossRefGoogle Scholar
  42. Suhasini K, Sagare AP, Krishnamurthy KV (1994) Direct somatic embryogenesis from mature embryo axes in chickpea (Cicerarietinum L.). Plant Sci 102:189–194CrossRefGoogle Scholar
  43. Thomas C, Bronner R, Molinier J, Prinsen E, van Onckelen H, Hahne G (2002) Immuno-cytochemical localization of indole-3-acetic acid during induction of somatic embryogenesis in cultured sunflower embryos. Planta 215:577–583Google Scholar
  44. Toonen MAJ, Hendriks T, Schmidt EDL, Verhoeve HA, Kammen A, De Vries SC (1994) Description of somatic-embryoforming single cells in carrot suspensions cultures employing video cell tracking. Planta 194:565–572CrossRefGoogle Scholar
  45. Valera-Montero LL, Ochoa-Alejo N (1992) A novel approach for chili pepper (Capsicum annuum L.) plant regeneration: shoot induction in rooted hypocotyls. Plant Sci 84:215–219CrossRefGoogle Scholar
  46. Venkataiah P, Christopher T, Subhash K (2003) Thiadiazuron induced high frequency adventitious shoot formation and plant regeneration in Capsicum annuum L. J Plant Biotechnol 5:245–250Google Scholar
  47. Williams EG, Maheswaran G (1986) Somatic embryogenesis: factors influencing coordinated behavior of cells as an embryogenic group. Ann Bot 57:443–462Google Scholar
  48. Xiao W, Custard KD, Brown RC, Lemmon BE, Harada JJ, Goldberg RB, Fischer RL (2006) DNA methylation is critical for Arabidopsis embryogenesis and seed viability. Plant Cell. 18:805–814Google Scholar
  49. Yeung EC, Rahman MH, Thorpe TA (1996) Comparative development of zygotic and microspore-derived embryos in Brassica napus L. cv. Topas. I. Histodifferentiation. Int J Plant Sci 157:27–39CrossRefGoogle Scholar
  50. Zapata-Castillo YP, Canto-Flick A, López-Puc G, Solís-Ruiz A, Barahona-Pérez F, Santana-Buzzy N (2007) Somatic Embryogenesis in Habanero Pepper (C. chinense Jacq.) from cell suspensions. HortScience 42(2):1–5Google Scholar
  51. Zuo J, Niu QW, Frugis G, Chua NH (2002) The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis. Plant J 30(3):349–359PubMedCrossRefGoogle Scholar

Copyright information

© Korean Society for Plant Biotechnology and Springer 2012

Authors and Affiliations

  • Susana A. Avilés-Viñas
    • 1
  • Carlos A. Lecona-Guzmán
    • 1
  • Adriana Canto-Flick
    • 1
  • Stephanie López-Erosa
    • 1
  • Nancy Santana-Buzzy
    • 1
    Email author
  1. 1.Unidad de Bioquímica y Biología Molecular de PlantasCentro de Investigación Científica de YucatánMéridaMexico

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