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Micropropagation of axillary shoots of hybrid chestnut (Castanea sativa × C. crenata) in liquid medium in a continuous immersion system


A protocol for culturing chestnut axillary shoots with the basal sections continuously immersed in liquid medium was developed and the influence of various parameters was assessed: explant type and size, support material, bioreactor size and aeration regime. Shoots excised from eight selected chestnut genotypes were successfully cultured in 10 L bioreactors in liquid Murashige and Skoog medium with half strength nitrates and supplemented with 0.05 mg/L N 6-benzyladenine and 30 g/L sucrose. Forced ventilation and the use of a support material were essential for producing healthy, non-hyperhydric shoots. High proliferation rates were achieved and good quality shoots ready for multiplication, rooting and acclimatization were obtained. Larger leaves showing higher levels of photosynthetic pigments were observed in shoots cultured in bioreactors, suggesting a certain degree of photoautotrophy in shoots cultured under forced ventilation. This is the first demonstration of the production of chestnut shoots in stationary liquid medium, indicating the feasibility of the method for large-scale propagation of the species.

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We acknowledge the contribution of our dear colleague and friend, Brais Bogo Graña, who died before completion of the manuscript. We thank Alejandro Díaz, Rafael Sánchez and Patricia Val for technical assistance. This research was partly funded through the FEDER INNTERCONECTA 2013/2014 programme (Project INTEGRACASTANEA EXP00064828/ITC-20133040).

Author information

BC, CS and NV conceived and designed the experiment. NV, AA, BB, BB and BC performed the experiments and analysed and interpreted the data. NV and CS wrote the manuscript. All authors read and approved the final manuscript.

Correspondence to N. Vidal.

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The authors declare that they have no conflict of interest.

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Communicated by Francisco de Assis Alves Mourão Filho.

Deceased: B. Bogo.

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Cuenca, B., Sánchez, C., Aldrey, A. et al. Micropropagation of axillary shoots of hybrid chestnut (Castanea sativa × C. crenata) in liquid medium in a continuous immersion system. Plant Cell Tiss Organ Cult 131, 307–320 (2017).

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  • Bioreactors
  • Castanea
  • Forced ventilation
  • Large-scale propagation