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Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta)

Abstract

This paper reports the first successful isolation and cell wall regeneration of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham protoplasts. These results form an important foundation for the development of a successful tissue culture system for G. gracilis. Initially, an isolation protocol was optimized by investigation of the effects of the enzyme constituents and concentrations, the pre-treatment of thalli, the incubation period and temperature, and the pH of the enzymatic medium on protoplast yields. A pre-treatment of G. gracilis thalli with 1 % (w/v) papain for 30 min followed by a 3-h enzymatic digestion of thalli with an enzymatic mixture containing 2 % (w/v) cellulase Onozuka R-10, 1 % (w/v) macerozyme R-10, and 10 U mL−1 agarase at pH 6.15 was found to produce the highest yield of protoplasts at 22 °C. Reliably high yields (20–30 × 105 protoplasts g−1 f.wt) of protoplasts could be obtained from G. gracilis thalli when this optimized protocol was used. Cell wall re-synthesis by G. gracilis protoplasts, which constitutes the first step towards whole plant regeneration, was followed using calcoflour staining and scanning electron microscopy. Protoplasts were shown to complete the initial stages of cell wall re-synthesis within the first 24 h of culturing.

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Acknowledgements

The authors thank Irvine and Johnson Abalone Culture Division, Danger Point, Gansbaai, South Africa for supplying G. gracilis and Miranda Waldron (EM Unit, University of Cape Town) for her assistance with the SEM. This work was funded by a National Research Foundation (NRF) grant (GUN: 2053564) awarded to VEC and a Medical Research Council of South Africa grant awarded to AEM. SMH was supported by a NRF Scarce Skills Scholarship and a University of Cape Town student bursary.

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Correspondence to Ann E. Meyers.

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Huddy, S.M., Meyers, A.E. & Coyne, V.E. Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta). J Appl Phycol 25, 433–443 (2013). https://doi.org/10.1007/s10811-012-9877-2

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  • DOI: https://doi.org/10.1007/s10811-012-9877-2

Keywords

  • Gracilaria
  • Protoplasts
  • Cell wall-degrading enzymes
  • Cell wall regeneration