Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts

  • S. V. Raikar
  • R. H. Braun
  • C. Bryant
  • A. J. Conner
  • M. C. Christey
Original Article


This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of micro-colonies with plating efficiencies 3–10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.


Feeder-layer Lotus corniculatus Nitrocellulose membrane Protoplast culture Protoplast isolation Shoot regeneration 


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Copyright information

© Korean Society for Plant Biotechnology and Springer 2008

Authors and Affiliations

  • S. V. Raikar
    • 1
    • 2
  • R. H. Braun
    • 2
  • C. Bryant
    • 3
  • A. J. Conner
    • 1
    • 2
  • M. C. Christey
    • 2
  1. 1.Bio-Protection and Ecology DivisionLincoln UniversityCanterburyNew Zealand
  2. 2.New Zealand Institute for Crop and Food Research LtdChristchurchNew Zealand
  3. 3.Pastoral Genomics LtdNewmarket, AucklandNew Zealand

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