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Background    Magnolia  is a woody ornamental plant, which is widely used in urban landscaping. However, its lengthy juvenile period and recalcitrance to regeneration impedes functional characterization of its genes.    Results  We developed an efficient protoplast isolation and transient expression system for  Magnolia denudata  ×  Magnolia acuminata  ‘Yellow River’. The highest yield of protoplasts was obtained from young leaves digested in 3% Cellulase R10, 0.8% Macerozyme R10, 0.04% pectinase and 0.4 M mannitol enzymolysis solution for 6 h. For transfection of protoplasts, 20% PEG4000 for 5 min was optimal. To verify the protoplast system and begin to understand heat tolerance in  Magnolia , a heat shock transcription factor  MdeHSF1  was cloned from ‘Yellow River’, which belongs to the HSF subfamily A and has significant homology with  AtHSFA1A . Subcellular localization analysis indicated that  MdeHSF1  was expressed in the cell nucleus. Furthermore, qPCR analysis of the  MdeHSF1  transcript level in response to high temperature stress suggested that  MdeHSF1  might be involved in regulating heat stress tolerance in ‘Yellow River’.    Conclusion  The described protocol provides a simple and straightforward method for isolating protoplast and exploring gene subcellular localization of  MdeHSF1  in  Magnolia . This expands the new research of protoplast isolation and transfection in  Magnolia .    Electronic supplementary material  The online version of this article (doi:10.1186/s13007-017-0193-3) contains supplementary material, which is available to authorized users.

Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1