Somatic Embryogenesis in Common BeanPhaseolus vulgaris L.

  • José Luis Cabrera-Ponce
  • Itzel Anayetzi González-Gómez
  • Claudia G. León-Ramírez
  • José A. Sánchez-Arreguín
  • Alba E. Jofre y Garfias
Part of the Methods in Molecular Biology book series (MIMB, volume 1815)


Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to induce somatic embryogenesis (SE) under in vitro conditions. An alternative strategy to yield SE is based upon the use of a cytokinin (benzyladenine) coupled with osmotic stress adaptation instead of the auxin-inducing SE in common bean. Here we described the induction of proembryogenic masses (PEM) derived from the apical meristem and cotyledonary zone of zygotic embryos, from which secondary SE indirect embryogenesis emerged. Maturation of SE was achieved by using osmotic stress medium and converted to plants. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and biobalistics as well as basic biochemical and molecular biology research.

Key words

Cytokinin Osmotic stress Phaseolus vulgaris Plant regeneration Somatic embryogenesis 



This work was supported in part by SAGARPA (Grant 2003-199). We thank Miss Sandra Chavez-León for her excellent technical assistance and Dra. Rosa Maria Rangel-Cano for the critical review of the manuscript.


  1. 1.
    Food and Agricultural Organization of the United Nations (FAO) (1999) PHASEOLUS BEAN: Post-harvest Operations. Organisation: Centro Internacional de Agricultura tropical (CIAT) Author: AL. Jones. Edited by AGSI/FAO: Danilo Mejia (Technical), Beverly Lewis (Language and style)
  2. 2.
    Veltcheva M, Svetleva D, Petkova SP, Perl A (2005) In vitro regeneration and genetic transformation of common bean (Phaseolus vulgaris L.)-problems and progress. Sci Hort 107:2–10. CrossRefGoogle Scholar
  3. 3.
    Delgado-Sánchez P, Saucedo-Ruiz M, Guzmán-Maldonado SH et al (2006) An organogenic plant regeneration system for common bean (Phaseolus vulgaris L.). Plant Sci 170:822–827. CrossRefGoogle Scholar
  4. 4.
    Arellano J, Fuentes SI, Castillo-España P, Hernández G (2009) Regeneration of different cultivars of common bean (Phaseolus vulgaris L.) via indirect organogenesis. Plant Cell Tiss Org 96:11–18. CrossRefGoogle Scholar
  5. 5.
    Gatica-Arias AM, Muñoz-Valverde J, Ramírez-Fonseca P, Valdez-Melara M (2010) In vitro plant regeneration system for common bean (Phaseolus vulgaris): effect of N6-benzylaminopurine and adenine sulphate. Electron J Biotechnol 13:a06. CrossRefGoogle Scholar
  6. 6.
    Kwapata K, Sabzikar R, Stcklen MB, Kelly JD (2010) In vitro regeneration and morphogenesis studies in common bean. Plant Cell Tissue Organ Cult 100:97–105. CrossRefGoogle Scholar
  7. 7.
    Collado R, Veitía N, Bermúdez-Caraballoso I et al (2013) Efficient in vitro plant regeneration via indirect organogenesis for different common bean cultivars. Sci Hort 153:109–116. CrossRefGoogle Scholar
  8. 8.
    Russell DR, Wallace KM, Bathe JH et al (1993) Stable transformation of Phaseolus vulgaris via electric-discharge mediated particle acceleration. Plant Cell Rep 12:165–169. CrossRefPubMedGoogle Scholar
  9. 9.
    Aragao FJL, Barros LMG, Brasileiro ACM et al (1996) Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment. Theor Appl Genet 93:142–150. CrossRefGoogle Scholar
  10. 10.
    Aragao FJL, Ribeiro SG, Barros LMG et al (1998) Transgenic beans (Phaseolus vulgaris L.) engineered to express viral antisense RNAs show delayed and attenuated symptoms to bean golden mosaic geminivirus. Mol Breed 4:491–499. CrossRefGoogle Scholar
  11. 11.
    Kim JW, Minamikawa T (1996) Transformation and regeneration of French bean plants by the particle bombardment process. Plant Sci 117:131–138. CrossRefGoogle Scholar
  12. 12.
    Bonfim K, Faria JC, Nogueira EOPL, Mendes EA, Aragao FJL (2007) RNAi-mediated resistance to bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol Plant Microbe In 20:717–726. CrossRefGoogle Scholar
  13. 13.
    Christou P (1990) Morphological description of transgenic soybean chimeras created by the delivery, integration and expression of foreign DNA using electric discharge particle acceleration. Ann Bot 66:379–386. CrossRefGoogle Scholar
  14. 14.
    Krastanova S, Perrin M, Barbier P et al (1995) Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus. Plant Cell Rep 13:357–360. CrossRefGoogle Scholar
  15. 15.
    Xiang YZ, Ying HS, Zhi JC, Xian SZ (2008) Cell fate switch during in vitro plant organogenesis. J Integ Plant Biol 50:816–824. CrossRefGoogle Scholar
  16. 16.
    Steward FC, Mapes MO, Smith J (1958) Growth and organized development of cultured cells. I. Growth and division of freely suspended cells. Am J Bot 45:693–703CrossRefGoogle Scholar
  17. 17.
    Haccius B (1978) Question of unicellular origin of non-zygotic embryos in callus cultures. Phytomorphology 28:74–81Google Scholar
  18. 18.
    Raghavan V (1976) Adventive embryogenesis: induction of diploid embryoids. In: Sutchliffe JF (ed) Experimental embryogenesis in vascular plants. Academic Press, London, pp 349–381Google Scholar
  19. 19.
    Xu Z, Zhang C, Zhang X et al (2013) Transcriptome profiling reveals auxin and cytokinin regulating somatic embryogenesis in different sister lines of cotton cultivar CCR124. J Integr Plant Biol 55:631–642. CrossRefPubMedGoogle Scholar
  20. 20.
    Allavena A, Rossetti L (1983) Efforts in somatic embryogenesis of Phaseolus vulgaris L. Acta Hort 131:239–246. CrossRefGoogle Scholar
  21. 21.
    Martins IS, Sondahl MR (1984) Early stages of somatic embryo differentiation from callus cells of bean (Phaseolus vulgaris L.) grown in liquid medium. J Plant Physiol 117:97–103. CrossRefPubMedGoogle Scholar
  22. 22.
    Saunders JW, Hosfield GL, Levi A (1987) Morphogenetic effects of 2,4-dichlorophenoxyacetic acid on pinto bean (Phaseolus vulgaris L.) leaf explants in vitro. Plant Cell Rep 6:46–49. CrossRefPubMedGoogle Scholar
  23. 23.
    Hoyos RA (1990) Somatic embryogenesis in common bean (Phaseolus vulgaris L.): influence of media and environmental factors on globular and mature embryoid formation. Department of Crop and Soil Science. Michigan State University, USAGoogle Scholar
  24. 24.
    Collado R, Garcia LR, Angenon G et al (2011) Somatic embryos formation from immature cotyledons in Phaseolus vulgaris cv. CIAP 7247. Biotechnol Veg 11:235–240Google Scholar
  25. 25.
    Malik KA, Saxena PK (1992) Somatic embryogenesis and shoot regeneration from intact seedlings of Phaseolus acutifolius A., P. aureus (L.) Wilczek, P. coccineus, and P. wrightii L. Plant Cell Rep 11:163–168. CrossRefPubMedGoogle Scholar
  26. 26.
    Garcia LR, Pérez J, Kosky RG et al (2010) Regeneración de plantas via embriogénesis somática en Phaseolus acutifolius A. Gray. Biotechnol Veg 10:143–149Google Scholar
  27. 27.
    Nafie EM, Taha HS, Mansur RM (2013) Impact of 24-epibrassinolide on callogenesis and regeneration via somatic embryogenesis in Phaseolus vulgaris L. cv Brunca. World Appl Sci J 24:188–200. CrossRefGoogle Scholar
  28. 28.
    Fehér A, Pasternak TP, Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant Cell Tiss Org 74:201–228. CrossRefGoogle Scholar
  29. 29.
    Karami O, Saidi A (2010) The molecular basis for stress-induced acquisition of somatic embryogenesis. Mol Biol Rep 37:2493–2507. CrossRefPubMedGoogle Scholar
  30. 30.
    Kamada H, Ishikawa K, Saga H, Harada H (1993) Induction of somatic embryogenesis in carrot by osmotic stress. Plant Tiss Cult Lett 10:38–44. CrossRefGoogle Scholar
  31. 31.
    Lou H, Kako S (1995) Role of high sugar concentrations in inducing somatic embryogenesis from cucumber cotyledons. Sci Hort 64:11–20. CrossRefGoogle Scholar
  32. 32.
    Ikeda-Iwai M, Umehara M, Satoh S, Kamada H (2003) Stress-induced somatic embryogenesis in vegetative tissues of Arabidopsis thaliana. Plant J 34:107–114. CrossRefPubMedGoogle Scholar
  33. 33.
    Karami O, Deljou A, Esna-Ashari M, Ostad-Ahmadi P (2006) Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophillus L.). Sci Hort 110:340–344. CrossRefGoogle Scholar
  34. 34.
    Dudits D., J. Györgyey, L. Bögre y L. Bakó, (1995) Molecular biology of somatic embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in plants, Kluwer Academic Publishers, Dordrecht, pp 267–308. doi: CrossRefGoogle Scholar
  35. 35.
    Werner T, Schmülling T (2009) Cytokinin action in plant development. Curr Opin Plant Biol 12:527–538. CrossRefPubMedGoogle Scholar
  36. 36.
    Ha S, Vankova R, Yamaguchi-Shinozaki K et al (2012) Cytokinins: metabolism and function in plant adaptation to environmental stresses. Trends Plant Sci 17:172–179. CrossRefPubMedGoogle Scholar
  37. 37.
    Cabrera-Ponce JL, Lopez L, León-Ramírez CG et al (2015) Stress induced acquisition of somatic embryogenesis in common bean Phaseolus vulgaris L. Protoplasma 252:559–570. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  39. 39.
    Barraza A, Cabrera-Ponce JL, Gamboa-Becerra R et al (2015) The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean. Front Plant Sci 6:577. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Kawashima T, Goldberg RB (2009) The suspensor: not just pending the embryo. Trends Plant Sci 15:23–30. CrossRefPubMedGoogle Scholar
  41. 41.
    Quiroz-Figueroa F, Rojas-Herrera R, Galaz-Avalos R, Loyola-Vargas V (2006) Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant Cell Tiss Org 86:285–301. CrossRefGoogle Scholar
  42. 42.
    Urao T, Yakubov B, Satoh R et al (1999) A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor. Plant Cell 11:1743–1754. CrossRefGoogle Scholar
  43. 43.
    Tran LSP, Urao T, Qin F et al (2007) Functional analysis of AHK1/ATHK1 and cytokinin receptor histidine kinases in response to abscisic acid, drought, and salt stress in Arabidopsis. Proc Natl Acad Sci U S A 104:20623–20628. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Jeon J, Kim YN, Kim S et al (2010) A subset of cytokinins two-component signaling system plays a role in cold temperature stress response in Arabidopsis. J Biol Chem 285:23371–23386CrossRefPubMedGoogle Scholar
  45. 45.
    Müller B, Sheen J (2008) Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature 453:1094–1097. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Hwang I, Sheen J (2001) Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413:383–389. CrossRefPubMedGoogle Scholar
  47. 47.
    Buechel S, Eibfried A, To JP et al (2010) Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration. Eur J Cell Biol 89:279–284. CrossRefPubMedGoogle Scholar
  48. 48.
    Su YH, Li YB, Bai B, Zhang XS (2015) Establishment of embryonic shoot-root axis in auxin and cytokinin response during Arabidopsis somatic embryogenesis. Front Plant Sci 5:792. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Murch SJ, KrishnaRaj S, Saxena PK (1997) Thidiazuron-induced morphogenesis of regal geranium (Pelargonium domesticum): a potential stress response. Physiol Plant 101:183–191. CrossRefGoogle Scholar
  50. 50.
    Victor JMR, Murthy BNS, Murch SJ et al (1999) Role of endogenous purine metabolism in thidiazuron induced somatic embryogenesis of peanut (Arachis hypogea L.). Plant Growth Regul 28:41–47. CrossRefGoogle Scholar
  51. 51.
    Ashihara H, Stasolla C, Loukanina N, Thorpe T (2001) Purine metabolism during white spruce somatic embryo development: salvage of adenine, adenosine and inosine. Plant Sci 160:647–657. CrossRefPubMedGoogle Scholar
  52. 52.
    Genga A, Allavena A (1991) Factors affecting morphogenesis from immature cotyledon of Phaseolus coccineus L. Plant Cell Tiss Org 27:189–196. CrossRefGoogle Scholar
  53. 53.
    El Dawayati MM, Abd El Bar OH, Zaid ZE, Zein El Din AFM (2012) In vitro morpho-histological studies of newly developed embryos from abnormal malformed embryos of date palms cv. Gundila under dessication effect of polyethelyne glycol treatments. Ann Agric Sci 57:117–128. CrossRefGoogle Scholar
  54. 54.
    Yeung EC, Brown DCW (1982) The osmotic environment of developing embryos of Phaseolus vulgaris. Z Pflanzenphysiol 106:149–156. CrossRefGoogle Scholar
  55. 55.
    Geerts P, Mergeai G, Baudoin JP (1999) Rescue of early-shaped embryos and plant regeneration of Phaseolus polyanthus Greenm. And Phaseolus vulgaris L. Biotechnol Agron Soc Environ 3:141–148Google Scholar
  56. 56.
    Roberts DR, Sutton BCS, Flinn BS (1990) Synchronous and high frequency germination of interior spruce somatic embryos following partial drying at high relative humidity. Can J Bot 68:1086–1090. CrossRefGoogle Scholar
  57. 57.
    Attree SM, Moore D, Sawhney VK, Fowke LC (1991) Enhanced maturation and desiccation tolerance of white spruce (Picea glauca (Moench) Voss) somatic embryos: effects of a non-plasmolysing water stress and abscisic acid. Ann Bot 68:519–525. CrossRefGoogle Scholar
  58. 58.
    Flinn BR, Roberts DR, Taylor IEP (1991) Evaluation of somatic embryos on interior spruce. Characterization and developmental regulation of storage proteins. Physiol Plant 82:624–632. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • José Luis Cabrera-Ponce
    • 1
  • Itzel Anayetzi González-Gómez
    • 1
  • Claudia G. León-Ramírez
    • 1
  • José A. Sánchez-Arreguín
    • 1
  • Alba E. Jofre y Garfias
    • 1
  1. 1.Departamento de Ingeniería Genética, Unidad IrapuatoCentro de Investigación y de Estudios Avanzados del IPN, CPGuanajuatoMexico

Personalised recommendations