Plant Growth Regulation

, Volume 38, Issue 3, pp 279–285 | Cite as

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala



Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN6-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l−1N6-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.

Callus Leucaena leucocephala Organogenesis Tissue culture 


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  1. Armstrong C.L. and Green C.E. 1985. Establishment and maintenance of friable, embryogenic maize callus and involvement of L-proline. Planta 164: 207–214.Google Scholar
  2. Brar M.S., Al-khayri J.M., Morelock T.E. and Anderson E.J. 1999. Genotypic response of Cowpea Vigna unguiculata (L.) to in vitro regeneration from cotyledon explants. In Vitro Cell. Dev. Biol.-Plant 35: 8–12.Google Scholar
  3. Buiteveld J., Fransz P.F. and Creemers-Molenaar J. 1994. Induction and characterization of embryogenic callus types for the initiation of suspension cultures of leek (Allium ampeloprasum L.). Plant Sci. 100: 195–202.Google Scholar
  4. Burnett L., Arnoldo M., Yarrow S. and Huang B. 1994. Enhancement of shoot regeneration from cotyledon explants of Brassica rapa spp oleifera through pretreatment with auxin and cytokinin and use of ethylene inhibitors. Plant Cell, Tiss. Organ Cult. 37: 253–256.Google Scholar
  5. Chang C.C., Moll C.B., Evenson K.B. and Guiltinan M.J. 1996. In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium. Plant Cell, Tiss. Organ Cult. 45: 61–66.Google Scholar
  6. Das P.K., hakravarti V. and Maity S. 1993. Plantlet formation in tissue culture from cotyledon of Acacia auriculiformis A. Cunn ex Benth. Ind. J. Forest. 16: 189–192.Google Scholar
  7. Gendy C., Sene M., Le B.V., Vidal J. and Van K.T.T. 1996. Somatic embryogenesis and plant regeneration in Sorghum bicolor (L.). Moench. Plant Cell Rep. 15: 900–904.Google Scholar
  8. Gill R. and Ozias-akins P. 1999. Thidiazuron-induced highly morphogenic callus and high frequency regeneration of fertile peanut (Arachis hypogaea L.) plants. In vitro Cell. Dev. Biol.-Plant 35: 445–450.Google Scholar
  9. Glovak L. and Greatbatch W. 1981. Successful tissue culture of Leucaena. Leucaena Res. Rep. 3: 81–82.Google Scholar
  10. Goyal Y., Bingham R.L. and Felker P. 1995. Progragation of tropical tree, Leucaena leucocephala K67, by in vitro bud culture. Cell Tissue Organ Cult. 2: 49–53.Google Scholar
  11. Jin R.-G., Liu Y.-B., Tabashnik B.E. and Borthakur D. 2000. Development of transgenic cabbage (Brassica oleracea var. capitata) for insect resistance by Agrobacterium tumefaciens-mediated transformation. In Vitro Cell. Dev. Biol.-Plant 36: 231–237.Google Scholar
  12. Jones R.J. 1979. The value of Leucaena leucocephala as a feed for ruminants in the tropics. World Animal Rev. 31: 13–23.Google Scholar
  13. Kuo Y.J. and Smith M.A.L. 1993. Plant regeneration from St. Augustine grass immature embryo-derived callus. Crop Sci. 33: 1394–1396.Google Scholar
  14. Lin H.-S., van der Toorn C., Raemakers K.J.J.M., Visser F., De Jeu M.J. and Jacobsen E. 2000. Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria. Plant Cell Rep. 19: 529–534.Google Scholar
  15. Mao A.A., Wetten A., Fay M.F. and Caligari P.D.S. 2000. In vitro propagation of Litsea cubeba (Lours.) Pers., a multipurpose tree. Plant Cell Rep. 19: 263–267.Google Scholar
  16. Murashige T. and Skoog F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15: 473–479.Google Scholar
  17. Nagl W., Ignacimuthu S. and Becker J. 1997. Genetic engineering and regeneration of Phaseolus and Vigna: State of the art and new attempts. J. Plant Physiol. 150: 625–644.Google Scholar
  18. Peaseley E.L. and Collins G.B. 1980. Development of an in vitro culture system for Leucaena. Leucaena Newslett. 1: 54.Google Scholar
  19. Sharma S.K. and Ramamurthy V. 2000. Micropropagation of 4-year-old elite Eucalyptus tereticornis trees. Plant Cell Rep. 19: 511–518.Google Scholar
  20. Shelton H.M. and Brewbaker J.L. 1994. Leucaena leucocephala-the most widely used forage three legume. In: Gutteridge R.C. and Shelton H.M. (eds), Forest Tree Legumes in Tropical Agriculture. CAB International, Wallingford, UK, pp. 15–30.Google Scholar
  21. Soedarjo M. and Borthakur D. 1996. Mimosine produced by the tree-legume Leucaena provides growth advantages to some Rhizobium strains that utilizes it as a source of carbon and nitrogen. Plant Soil 186: 87–92.Google Scholar
  22. Sunyal M., Gupta S.D., Jana M.K. and Kundu S.C. 1998. Shoot organogenesis and plant regeneration from leaf callus culture of Tuberose (Polianthes tuberosa L.). Plant Tiss. Cult. Biotechnol. 4: 81–86.Google Scholar
  23. Thomas T.D., Bhatnagar A.K. and Bhojiwani S.S. 2000. Production of triploid plants of mulberry (Morus alba L.) by endosperm culture. Plant Cell Rep. 19: 395–399.Google Scholar
  24. Tuskan G.A., Sargent W.A., Rensema T. and Walla J.A. 1990. Influence of plant growth regulators, basal media and carbohydrate levels on the in vitro development of Pinus ponderosa (Dougl. ex Law) cotyledon explants. Plant Cell, Tiss. Organ Cult. 20: 47–52.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  1. 1.Department of Molecular Biosciences and BioengineeringUniversity of HawaiiHonoluluUSA

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