Efficient isolation of protoplasts and the application to subcellular localization of .

Background: 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  ×  '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 , a heat shock transcription factor was cloned from 'Yellow River', which belongs to the HSF subfamily A and has significant homology with . Subcellular localization analysis indicated that was expressed in the cell nucleus. Furthermore, qPCR analysis of the transcript level in response to high temperature stress suggested that 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 in . This expands the new research of protoplast isolation and transfection in .