Callus induction and plantlet regeneration in Bixa oreliana L., an annatto-yielding tree

  • P. S. Sha Valli Khan
  • E. Prakash
  • K. R. Rao


A protocol has been developed for plantlet regeneration from seed callus of Bixa orellana L. Seeds demonstrated a high percentage of callus induction (63±7.3%) and a high yield (356±14.7 mg per seed) of white friable callus on Murashige and Skoog (MS) medium containing 5.0 μM l-naphthaleneacetic acid (NAA) and 2.5μM N 6-benzyladenine (BA) within 6 wk of culture in the dark. Callus induction frequency was greater under 24h dark as compared to 16h light/8h dark photoperiod or 24h light photoperiod. Increased myo-inositol (MI: 200mgl−1) and addition of ascorbic acid (AA: 200 mgl−1) to the culture medium positively improved callus induction frequency and growth. Shoot differentiation from white friable seed callus was best using 10.0 μM BA and 5.0 μM NAA, where the highest percentage of calluses forming shools (74.9±4.8%), the highest number of shoots per callus (six or seven) and the highest shoot-forming index (5.0) were obtained within 6 wk. Shoots elongated to 4 cm within 4 wk of transfer onto MS medium devoid of growth regulators. Shoots were rooted using half-strength MS medium containing 5.0 μM indole-3-butyric acid (IBA). About 85% of these plants were established in pots containing pure garden soil and organic manure after 3 wk of hardening. Regenerated plants were morphologically uniform with normal leaf, shape and growth patterns. These plants are currently being screened for the presence of agronomically useful genetic variants.

Key words

annatto Bixa orellana Bixaceae plantlet regeneration seed callus 


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  1. Ajitkumar, D.; Seemi, S. Rapid clonal multiplication through in vitro axillary shoot proliferation of Aegle marmelos (L.) Corr., a medicinal tree. Plant Cell Rep. 17:422–426; 1998.CrossRefGoogle Scholar
  2. Bennici, A.; Grifoni, T.; Schiff, S.; Bovelli, R. Studies on callus growth and morphogenesis in several species and lines of Amaranthus. Plant Cell Tiss. Organ Cult. 49:29–33; 1997.CrossRefGoogle Scholar
  3. Bhat, S. R.; Kackar, A.; Vieitez, A. M. In vitro adventitious bud regeneration from internode segments of beech. Plant Cell Tiss. Organ Cult. 60:213–220; 2000.CrossRefGoogle Scholar
  4. de Feo, V. Medicinal and magical plants in the northern Peruvian Andes. Fitoterapia 63:417–440; 1992.Google Scholar
  5. Eira, M. T. S.; Mello, C. M. C. Bixa orellana L. seed germination and conservation. Seed Sci. Tech. 25:373–380; 1997.Google Scholar
  6. Joy, R. W.; Patel, K. R.; Thorpe, T. A. Ascorbic acid enhancement of organogenesis in tobacco callus. Plant Cell Tiss. Organ Cult. 13:219–223; 1988.CrossRefGoogle Scholar
  7. Larkin P. J.; Scowcroft, W. R. Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60:197–214; 1981.CrossRefGoogle Scholar
  8. Lin, G. D.; Griffin, W. J. Organogenesis and a general procedure for plant regeneration from callus culture of a commercial Dubosia hybrid (D. Leichhardtii × D. myoporoides). Plant Cell Rep. 11:207–210; 1992.CrossRefGoogle Scholar
  9. Liu, W.; Chilcott, C. E.; Reich, R.; Hellmann, G. M. Regeneration of Salvia sclarea via organogenesis. In Vitro Cell Dev. Biol. Plant. 36:201–206; 2000.Google Scholar
  10. Loreti, F.; Muleo, R.; Morini, S. Effect of light quality on growth of in vitro cultured organs and tissues. Comb. Proc. Int. Plant Prop. Soc. 40:615–623; 1991.Google Scholar
  11. Mercadante, A. Z.; Steck, A.; Pfander, H. Isolation and identification of new apocarteniods from annatto (Bixa orellana) seeds. J. Agric. Food Chem. 45:1050–1054; 1997.CrossRefGoogle Scholar
  12. Millam, S.; Mitchell, S. M.; Moscheni, E.; Lyon, J. E. The establishment and regeneration of a range of Cuphea germplasm in vitro. Plant Cell Tiss. Organ Cult. 48:143–146; 1997.CrossRefGoogle Scholar
  13. Mondal, M.; Gupta, S.; Mukherjee, B. B. Callus culture and plantlet production in Carica papaya (Var. Honey Dew). Plant Cell Rep. 13:390–393; 1994.CrossRefGoogle Scholar
  14. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497; 1962.CrossRefGoogle Scholar
  15. Peterson, K. K. K. Callus induction and plant regeneration in Miscanthus × ogiformis Honda ‘Gigantcus’ as influenced by benzyladenine. Plant Cell Tiss. Organ Cult. 49:137–140; 1997.CrossRefGoogle Scholar
  16. Prakash, E.; Sha Valli Khan, P. S.; Sai Ram Reddy, P.; Rao, K. R. Regeneration of plants from seed-derived callus of Hybanthus enneaspermus L. Muell., a rare ethanobotanical herb. Plant Cell Rep. 18:873–878; 1999.CrossRefGoogle Scholar
  17. Saborio, F.; Dvorak, W.; Donahue, J. K.; Thorpe, T. A. In vitro regeneration of plantlets from mature embryos of Pinus ayacahuite. Tree Physiol. 17:787–796; 1997.PubMedGoogle Scholar
  18. Scotter, M. J.; Wilson, L. A.; Appleton, G. P.; Castle, L. Analysis of annatto (Bixa orellana) food coloring formulations. 1. Determination of coloring components and colored thermal degradation products by high-performance liquid chromatography with photodiode array detection. J. Agric. Food Chem. 46:1031–1038; 1998.CrossRefGoogle Scholar
  19. Siril, E. A.; Dhar, U. A highly efficient in vitro regeneration methodology for mature Chinese tallow tree (Sapium sebiferum, Roxb.). Plant Cell Rep. 16:83–87; 1996.Google Scholar
  20. Staudt, G. The effect of myo-inositol on the growth of callus tissue in Vitis. J. Plant Physiol. 116:161–166; 1984.Google Scholar
  21. Vengadesan, G.; Ganapathi, A.; Prem Anand, R.; Anbazhagan, V. R. In vitro organogenesis and plant formation in Acacia sinuta. Plant Cell Tiss. Organ Cult. 61:23–28; 2000.CrossRefGoogle Scholar
  22. Whitaker, R. J.; Burlington, N. J. Albienol-producing somaclonal variants of Nicotiana. US Patent no. 5, 117, 306; 1993.Google Scholar

Copyright information

© Society for In Vitro Biology 2002

Authors and Affiliations

  1. 1.Plant Tissue Culture, LaboratoryTata Energy Research InstituteNew DelhiIndia
  2. 2.Plant Tissue Culture Laboratory, Department of BotanySri Venkateswara UniversityTirupatiIndia

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