Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 106, Issue 3, pp 409–418 | Cite as

Somatic embryogenesis in the medicinal legume Desmodium motorium (Houtt.) Merr.

  • B. Chitra Devi
  • V. Narmathabai
Original Paper


An efficient protocol was established for regeneration of Desmodium motorium via somatic embryogenesis. Embryogenic calli were induced from cotyledon segments (6 mm, 16 days old) lacking embryo axis, excised from seedlings grown in vitro on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (2.9 μM) in combination with 6-benzyladenine (BA) (4.44 and 8.88 μM). Differentiation of embryogenic calli into globular and heart-shaped somatic embryos was achieved on transfer to hormone-free MS medium. When incubated for 4 days on MS medium supplemented with BA (8.88 μM), 95% of the globular and heart-shaped somatic embryos matured into torpedo and cotyledonary stages with minimum (10%) abnormalities. Modified MS basal medium without hormones and containing half-strength macronutrients and 0.88 M sucrose was suitable for germination of mature somatic embryos. Regenerated plantlets were successfully transferred to earthen pots with survival rate of 50%. Secondary embryogenesis was observed when pre-existing somatic embryos at globular and heart-shaped stages were cultured on MS medium supplemented with various concentrations of BA, adenine sulphate (AdS) and abscisic acid (ABA) individually.


Somatic embryogenesis Fabaceae Cotyledon explants Secondary embryogenesis 



2,4-Dichlorophenoxyacetic acid


Abscisic acid


Adenine sulphate




Indole-3-acetic acid


Murashige and Skoog (1962) medium


α-Naphthalene acetic acid


  1. Agrawal V, Sardar PR (2007) In vitro regeneration through somatic embryogenesis and organogenesis using cotyledons of Cassia angustifolia Vahl. In Vitro Cell Dev Biol-Plant 43:585–592CrossRefGoogle Scholar
  2. Ahmed R, Gupta SD, De DN (1996) Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean (Psophocarpus tetragonolobus L.). Plant Cell Rep 15:531–535Google Scholar
  3. Ammirato PV (1983a) Embryogenesis. In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook of plant cell culture, Vol I. Techniques for propagation and breeding, MacMillan, New YorkGoogle Scholar
  4. Ammirato PV (1983b) The regulation of somatic embryo development in plant cell culture: suspension culture technique and hormone requirement. Biotechnol 1:68–74CrossRefGoogle Scholar
  5. Bakshi DNG, Sensarma P, Pal DC (2001) A lexicon of medicinal plants in India. vol 2. Naya Prakash, Calcutta, pp 52–53Google Scholar
  6. Barua KN, Barua IC, Das M (2003) Ethnobotany of Rajbanshis of Assam. In: Maheshwari JK (ed) Ethnobotany and medicinal plants of Indian subcontinent. Scientific, Jodhpur, p 609Google Scholar
  7. Barwale UB, Kerns HR, Wildhom JM (1986) Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis. Planta 164:473–481CrossRefGoogle Scholar
  8. Buchheim JA, Colburn SM, Ranch JP (1989) Maturation of soybean somatic embryos and the transition to plantlet growth. Plant Physiol 89:768–775PubMedCrossRefGoogle Scholar
  9. Burns JA, Wetzstein HY (1998) Embryogenic cultures of the leguminous tree Albizia julibrissin and recovery of plants. Plant Cell Tiss Org Cult 54:55–59CrossRefGoogle Scholar
  10. Canhoto JM, Rama SC, Cruz GS (2006) Somatic embryogenesis and plant regeneration in carob (Ceratonia siliqua L.). In Vitro Cell Dev Biol-Plant 42:514–519CrossRefGoogle Scholar
  11. Chai M, Jia Y, Chen S, Gao Z, Wang H et al (2011) Callus induction, plant regeneration, and long-term maintenance of embryogenic cultures in Zoysia matrella [L.] Merr. Plant Cell Tiss Org Cult 104:187–192CrossRefGoogle Scholar
  12. Chen AH, Yang JL, Niu YD, Yang CP, Liu GF et al (2010) High-frequency somatic embryogenesis from germinated zygotic embryos of Schisandra chinensis and evaluation of the effects of medium strength, sucrose, GA3, and BA on somatic embryo development. Plant Cell Tiss Org Cult 102:357–364CrossRefGoogle Scholar
  13. Cucco MF, Jaume ADR (2000) Protocol for regeneration in vitro of Arachis hypogaea L. Electron J Biotechnol 3:154–160Google Scholar
  14. Das N, Bhadoria PBS (2006) Traditional knowledge and indigenous uses of tribal medicinal resources in India-A review. In: Govil JN, Singh VK, Arunachalam C (eds) Recent progress in medicinal plants, vol 13. Search for natural drugs. Studium, USA, pp 1–48Google Scholar
  15. Droste A, Leite PCP, Pasquali G, Mundstock ECD, Zanettini MHB (2001) Regeneration of soybean via embryogenic suspension culture. Scientia Agricola 58:753–758CrossRefGoogle Scholar
  16. Fernando JA, Vieira MLC, Geraldi IO, Gloria BAD (2002) Anatomical study of somatic embryogenesis in Glycine max (L.) Merrill. Brasil Archives Biol Technol 45:277–286Google Scholar
  17. Finer JJ, Nagasawa A (1988) Development of an embryogenic suspension culture of soybean (Glycine max Merril.). Plant Cell Tiss Org Cult 15:125–136CrossRefGoogle Scholar
  18. Gallego P, Hita O, Villalobos N, Dorado A, Martin L, Guerra H (2001) Somatic embryogenesis and plant regeneration in Medicago arborea L. In Vitro Cell Dev Biol-Plant 37:199–203CrossRefGoogle Scholar
  19. Garg L, Bhandari NN, Rani V, Bhojwani SS (1996) Somatic embryogenesis and regeneration of triploid plants in endosperm cultures of Acacia nilotica. Plant Cell Rep 15:855–858CrossRefGoogle Scholar
  20. Genga A, Allavena A (1991) Factors affecting morphogenesis from immature cotyledons of Phaseolus coccineus L. Plant Cell Tiss Org Cult 27:189–196CrossRefGoogle Scholar
  21. Ghanti KS, Rao S, Venugopal RB, Rao SR, Kaviraj CP, Barad A (2002) Somatic embryogenesis and Agrobacterium- mediated transformation in chickpea (Cicer arietinum L.). Plant Cell Biotechnol Mol Biol 3:51–58Google Scholar
  22. Ghosal S, Mazumder UK, Mehta R (1972) Indole bases of Desmodium gyrans. Phytochemistry 11:1863–1864CrossRefGoogle Scholar
  23. Girach RD, Aminuddin BS, Singh VK, Siddiqui MK (2007) Ethnomedicinal plants in skin care from Orissa, India. In: Govil JN, Singh VK, Singh S (eds) Recent progress in medicinal plants, vol 2. Phytochemistry and pharmacology. Studium, USA, pp 11–78Google Scholar
  24. Girija S, Ganapathi A, Ananthakrishnan G (2000) Somatic embryogenesis in Vigna radiata (L.) Wilczek. Ind J Exp Biol 38:1241–1244Google Scholar
  25. Gray DJ, Purohit A (1991) Somatic embryogenesis and development of synthetic seed technology. Crit Rev Plant Sci 10:33–61CrossRefGoogle Scholar
  26. Hiraga S, Minakawa H, Takahashi K, Takahashi R, Hajika M, Harada K, Ohtsubo N (2007) Evaluation of somatic embryogenesis from immature cotyledons of Japanese soybean cultivars. Plant Biotechnol 24:435–440CrossRefGoogle Scholar
  27. Hita O, Lafarga C, Guerra H (1997) Somatic embryogenesis from chickpea (Cicer arietinum L.) immature cotyledons: the effect of zeatin, gibberellic acid and indole-3-butyric acid. Acta Physiol Plant 19:333–338CrossRefGoogle Scholar
  28. Hita O, Gallego P, Villalobos N, Lanas I, Blazquez A, Martin JP, Fernandez J, Martin L, Guerra H (2003) Improvement of somatic embryogenesis in Medicago arborea. Plant Cell Tiss Org Cult 72:13–18CrossRefGoogle Scholar
  29. Hofmann N, Nelson RL, Korban SS (2004) Influence of media components and pH on somatic embryo induction in three genotypes of soybean. Plant Cell Tiss Org Cult 77:157–163CrossRefGoogle Scholar
  30. Iantcheva A, Vlahova M, Bakalova E, Kondorosi E, Elliott MC, Atanassov A (1999) Regeneration of diploid annual medics via direct somatic embryogenesis promoted by thidiazuron and benzylaminopurine. Plant Cell Rep 18:904–910CrossRefGoogle Scholar
  31. Iantcheva A, Vlahova M, Trinh TH, Brown SC, Slater A, Elliot MC, Atanassov A (2001) Assessment of polysomaty, embryo formation and regeneration in liquid media for various species of diploid annual Medicago. Plant Sci 160:621–627PubMedCrossRefGoogle Scholar
  32. Iantcheva A, Slavov S, Prinsen E, Vlahova M, Onckelen HV, Atanassov A (2005a) Embryo induction and regeneration from root explants of Medicago truncatula after osmotic pre-treatment. Plant Cell Tiss Org Cult 81:37–43CrossRefGoogle Scholar
  33. Iantcheva A, Vlahova M, Gvetoslavova S, Evtimova M, Atanassov A (2005b) Somatic embryogenesis of the model legume- Medicago truncatula and other diploid medics. Biotechnol & Biotechnol Eq 19:41–47Google Scholar
  34. Jain SK (1991) Dictionary of Indian folk medicine and ethnobotany-a reference manual of man-plant relationships, ethnic groups and ethnobotanists in India. Deep, India, p 72Google Scholar
  35. Jimenez VM (2001) Regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones. R Bras Fisiol Veg 13:196–223CrossRefGoogle Scholar
  36. Kaviraj CP, Kiran G, Venugopal RB, Kishor PBK, Rao S (2006) Somatic embryogenesis and plant regeneration from cotyledonary explants of green gram [Vigna radiata (L.) Wilczek.]- a recalcitrant grain legume. In Vitro Cell Dev Biol-Plant 42:134–138Google Scholar
  37. Komatsuda T, Lee W, Oka S (1992) Maturation and germination of somatic embryos as affected by sucrose and plant growth regulators in soybeans Glycine gracilis Skvortz and Glycine max (L.) Merr. Plant Cell Tiss Org Cult 28:103–113CrossRefGoogle Scholar
  38. Korbes AP, Droste A (2005) Carbon sources and polyethylene glycol on soybean somatic embryo conversion. Pesq Agropec Bras, Brasilia 40:211–216CrossRefGoogle Scholar
  39. Kumar VD, Kirti PB, Sachan JKS, Chopra VL (1995) Picloram induced somatic embryogenesis in chickpea (Cicer arietinum L.). Plant Sci 109:207–213Google Scholar
  40. Kumar AS, Gamborg OL, Nabors MW (1988) Plant regeneration from cell suspension cultures of Vigna aconitifolia. Plant Cell Rep 7:138–144CrossRefGoogle Scholar
  41. Kumar NA, Sivadasan M, Ravi N (2005) Flora of Pathanamthitta (Western Ghats, Kerala, India). Daya, Delhi, p 175Google Scholar
  42. Lakshmanan P, Taji A (2000) Somatic embryogenesis in leguminous plants. Plant Biol 2:136–148CrossRefGoogle Scholar
  43. Lalramnghinglova H (2003) Ethnomedicinal Plants of Mizoram. Bishen Singh Mahendra Pal Singh, Dehra Dun, p 135Google Scholar
  44. Li J, Grabau E (1996) Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars. Plant Cell Tiss Org Cult 44:87–89CrossRefGoogle Scholar
  45. Liao YK, Jin WR, Huang MC (2005) Pharmacognostic studies on Desmodium gyrans. Zhongguo Zhong Yao Za Zhi 30:175–177PubMedGoogle Scholar
  46. Liu CM, Xu ZH, Chua NH (1993) Auxin polar transport is essential for the establishment of bilateral symmetry during early plant embryogenesis. Plant Cell 5:621–630PubMedCrossRefGoogle Scholar
  47. Lou JP, Jia JF, Gu YH, Liu J (1999) High frequency somatic embryogenesis and plant regeneration in callus cultures of Astragalus adsurgens Pall. Plant Sci 143:93–99CrossRefGoogle Scholar
  48. Maheshwaran G, Williams EG (1986) Direct secondary somatic embryogenesis from immature sexual embryos of Trifolium repens cultured in vitro. Ann Bot 57:109–117Google Scholar
  49. Malik KA, Saxena PK (1992) Regeneration of Phaseolus vulgaris L.: high frequency induction of direct shoot formation in intact seedling by N6-benzlyaminopurine and thidiazuron. Planta 186:384–389CrossRefGoogle Scholar
  50. Martin AB, Cuadrado Y, Guerra H, Gallego P, Hita O, Martin L, Dorado A, Villalobos N (2000) Differences in the content of total sugars, reducing sugars, starch and sucrose in embryogenic and non-embryogenic calli from Medicago arborea L. Plant Sci 154:143–151PubMedCrossRefGoogle Scholar
  51. Maunder M, Higgens S, Culham A (1998) Neither common nor garden: the garden as a refuge for threatened plant species. Curtis’s Bot Mag 15:124–132CrossRefGoogle Scholar
  52. Merkle SA, Parrott WA, Flinn BS (1995) Morphogenic aspects of somatic embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer Academic, Dordrecht, pp 155–203Google Scholar
  53. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  54. Nolan KE, Rose RJ, Gorst JR (1989) Regeneration of Medicago truncatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants. Plant Cell Rep 8:278–281CrossRefGoogle Scholar
  55. Orczyk AN (1992) Somatic embryogenesis of agriculturally important lupin species (Lupinus angustifolius, L. albus, L. mutabilis). Plant Cell Tiss Org Cult 28:19–25CrossRefGoogle Scholar
  56. Ortiz BOC, Reyes MEP, Balch EPM (2000) Somatic embryogenesis and plant regeneration in Acacia farnesiana and A. schaffneri. In Vitro Cell Dev Biol-Plant 36:268–272CrossRefGoogle Scholar
  57. Pacheco G, Gagliardi RF, Valls JFM, Mansur E (2009) Micropropagation and in vitro conservation of wild Arachis species. Plant Cell Tiss Org Cult 99:239–249CrossRefGoogle Scholar
  58. Palmer CD, Keller WA (2010) Plant regeneration using immature zygotic embryos of Tribulus terrestris. Plant Cell Tiss Org Cult. doi:  10.1007/s11240-010-9838-x
  59. Pan Z, Zhu S, Guan R, Deng X (2010) Identification of 2, 4-D-responsive proteins in embryogenic callus of Valencia sweet orange (Citrus sinensis Osbeck) following osmotic stress. Plant Cell Tiss Org Cult 103:145–153CrossRefGoogle Scholar
  60. Panda H (2004) Handbook on herbal drugs and its plant sources. National Institute of Industrial Research, Delhi, p 410Google Scholar
  61. Parrott WA, Durham RE, Bailey MA (1995) Somatic embryogenesis in legumes. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 31. Somatic embryogenesis and synthetic seed II. Springer, BerlinGoogle Scholar
  62. Prange ANS, Serek M, Bartsch M, Winkelmann T (2010) Efficient and stable regeneration from protoplasts of Cyclamen coum Miller via somatic embryogenesis. Plant Cell Tissue Organ Cult 101:171–182CrossRefGoogle Scholar
  63. Pullaiah T (2002) Medicinal plants in India, vol 1. Regency, New Delhi, pp 215–216Google Scholar
  64. Rajasekaran K, Pellow JW (1997) Somatic embryogenesis from cultured epicotyls and primary leaves of soybean [Glycine max (L.) Merrill]. In Vitro Cell Dev Biol-Plant 33:88–91Google Scholar
  65. Ram M, Prasad KV, Kaur C, Singh SK, Arora A et al (2011) Induction of anthocyanin pigments in callus cultures of Rosa hybrida L. in response to sucrose and ammonical nitrogen levels. Plant Cell Tiss Org Cult 104:171–179CrossRefGoogle Scholar
  66. Rao MM, Lakshmisita G (1996) Direct somatic embryogenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.). Plant Cell Rep 15:355–359CrossRefGoogle Scholar
  67. Rout GR, Samantaray S, Das P (1995) Somatic embryogenesis and plant regeneration from callus cultures of Acacia catechu-a multipurpose leguminous tree. Plant Cell Tiss Org Cult 42:283–285CrossRefGoogle Scholar
  68. Santos AVP, Cutter EG, Davey MR (1983) Origin and development of somatic embryos in Medicago sativa L. (alfalfa). Protoplasma 117:107–115CrossRefGoogle Scholar
  69. Santos KGBD, Mariath JEDA, Moco MCC, Zanettini MHB (2006) Somatic embryogenesis from immature cotyledons of soybean [Glycine max (L.) Merr.]: Ontogeny of somatic embryos. Brasil Archives Biol Technol 49:49–55Google Scholar
  70. Sarasan V, Cripps R, Ransay MM, Atherton C, McMichen M, Predergast G, Rowntree JK (2006) Conservation in vitro of threatened plants-progress in the past decade. In Vitro Cell Dev Biol-Plant 42:206–214CrossRefGoogle Scholar
  71. Sen J, Kalia S, Mukherjee SG (2002) Level of endogenous free amino acids during various stages of culture of Vigna mungo (L.) Hepper- somatic embryogenesis, organogenesis and plant regeneration. Curr Sci 82:429–433Google Scholar
  72. Shekhawat NS, Galston AW (1983) Isolation, culture and regeneration of moth bean (Vigna aconitifolia) leaf protoplasts. Plant Sci Lett 32:43–51CrossRefGoogle Scholar
  73. Sita GL, Rao MM (2005) Direct somatic embryogenesis from immature seeds of rosewood (Dalbergia latifolia Roxb.). In: Pareek LK, Swarnkar PL (eds) Trends in plant tissue culture and biotechnology. Agrobios, Jodhpur, pp 271–279Google Scholar
  74. Sreenivasu K, Malik SK, Kumar PA, Sharma RP (1998) Plant regeneration via somatic embryogenesis in pigeonpea (Cajanus cajan L. Millsp). Plant Cell Rep 17:294–297CrossRefGoogle Scholar
  75. Street HE (1979) Embryogenesis and chemically induced organogenesis. In: Sharp WR, Larsen PO, Paddock EF, Raghavan V (eds) Plant cell and tissue culture–principles and application. Ohio State University Press, Columbus, pp 127–153Google Scholar
  76. Stuart DA, Nelsen J, McCall CM, Strickland SG, Walker KA (1985) Physiology of the development of somatic embryos in cell cultures of alfalfa and celery. In: Zaitlin M, Day P, Hollaender A, Wilson CM (eds) Biotechnology in plant science, relevance to agriculture in the eighties. Academic, Florida, pp 35–178Google Scholar
  77. Sukumaran S, Jeeva S, Raj DAS, Kannan D (2008) Floristic Diversity, conservation status and economic value of miniature sacred groves in Kanyakumari district, Tamil Nadu, Southern Peninsular India. Turk J Bot 32:185–199Google Scholar
  78. Toonen MAJ, Hendriks T, Schmidt EDL, Verhoeven HA, Kammen AV, Vries SCD (1994) Description of somatic embryo-forming single cells in carrot suspension cultures employing video cell tracking. Planta 194:565–572CrossRefGoogle Scholar
  79. Trout K (2004) Trout’s notes on the genus Desmodium, chemistry, ethnomedicine, pharmacology, synonyms and miscellany. Better Days, USAGoogle Scholar
  80. Venkatachalam P, Geetha N, Khandelwal A, Shaila MS, Sita GL (1999) Induction of direct somatic embryogenesis and plant regeneration from mature cotyledon explants of Arachis hypogaea L. Curr Sci 77:269–272Google Scholar
  81. Viba JB, Choudhary K, Singh M, Rathore MS, Shekhawat NS (2009) An efficient somatic embryogenesis system for velvet bean [Mucuna pruriens (L.) DC.]: a source of anti Parkinson’s drug. Plant Cell Tiss Org Cult 99:319–325CrossRefGoogle Scholar
  82. Walker DR, Parrott WA (2001) Effect of polyethylene glycol and sugar alcohols on soybean somatic embryo germination and conversion. Plant Cell Tiss Org Cult 64:55–62CrossRefGoogle Scholar

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© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Department of BiotechnologyKarpagam UniversityCoimbatoreIndia
  2. 2.Department of BotanyBharathiar UniversityCoimbatoreIndia

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