Abstract
BackgroundMagnolia 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.ResultsWe 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’.ConclusionThe 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.
Highlights
Magnolia is a woody ornamental plant, which is widely used in urban landscaping
Protoplast isolation and transformation was performed as previously described, and the MdeHSF1 transient expression vector was successfully expressed in the ‘Yellow River’ protoplast (Fig. 7a). These results confirmed that MdeHSF1 was expressed in the cell nucleus (Fig. 7b), and demonstrated the utility of the protoplast isolation and transient expression system we developed
[33], Oryza sativa [16, 34], Malus × domestica [35] and Fragaria × ananassa [36]. These protoplast transient expression systems have been used in a variety of experimental studies and have facilitated the advancement of gene function research [16, 34] In contrast, there are a limited number of reports on protoplast isolation and transient expression in ornamental plants
Summary
Magnolia is a woody ornamental plant, which is widely used in urban landscaping. Its lengthy juvenile period and recalcitrance to regeneration impedes functional characterization of its genes. Magnolia is among the best known ornamental plant used in landscaping. The majority of Magnolia species are distributed in America and East Asia, including relict plants, some of which have survived for hundreds of millions of years [1]. Many species of Magnolia growing in the wild are endangered, and some face extinction. Several factors have contributed to this situation, including threats to their habitats, limited genetic diversity, and difficulties associated with breeding. Efforts are being made to protect, develop, and spread magnolias. Grafting and seeding are the main methods for propagating Magnolia.
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