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Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts

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Abstract

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.

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Acknowledgments

The authors thank The Foundation for Research Science and Technology New Zealand and Pastoral Genomics Ltd., Auckland New Zealand for supporting S.V.R. through an Enterprise Scholarship. S.V.R. also gratefully acknowledges the support provided by Lincoln University in the form of a ‘Writing Scholarship’.

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Authors and Affiliations

  1. Bio-Protection and Ecology Division, Lincoln University, P.O. Box 84, Canterbury, New Zealand

    S. V. Raikar & A. J. Conner

  2. New Zealand Institute for Crop and Food Research Ltd, Private Bag 4704, Christchurch, 8140, New Zealand

    S. V. Raikar, R. H. Braun, A. J. Conner & M. C. Christey

  3. Pastoral Genomics Ltd, P.O. Box 109-185, Newmarket, Auckland, New Zealand

    C. Bryant

Authors
  1. S. V. Raikar
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  2. R. H. Braun
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  3. C. Bryant
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  4. A. J. Conner
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  5. M. C. Christey
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Correspondence to M. C. Christey.

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Raikar, S.V., Braun, R.H., Bryant, C. et al. Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts. Plant Biotechnol Rep 2, 171–177 (2008). https://doi.org/10.1007/s11816-008-0058-3

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  • DOI: https://doi.org/10.1007/s11816-008-0058-3

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