Abstract
BackgroundPlant protoplasts constitute unique single-cell systems that can be subjected to genomic, proteomic, and metabolomic analysis. An effective and sustainable method for preparing protoplasts from tea plants has yet to be established. The protoplasts were osmotically isolated, and the isolation and purification procedures were optimized. Various potential factors affecting protoplast preparation, including enzymatic composition and type, enzymatic hydrolysis duration, mannitol concentration in the enzyme solution, and iodixanol concentration, were evaluated.ResultsThe optimal conditions were 1.5% (w/v) cellulase and 0.4–0.6% (w/v) macerozyme in a solution containing 0.4 M mannitol, enzymatic hydrolysis over 10 h, and an iodixanol concentration of 65%. The highest protoplast yield was 3.27 × 106 protoplasts g−1 fresh weight. As determined through fluorescein diacetate staining, maximal cell viability was 92.94%. The isolated protoplasts were round and regularly shaped without agglomeration, and they were less than 20 μm in diameter. Differences in preparation, with regard to yield and viability in the tissues (roots, branches, and leaves), cultivars, and cultivation method, were also observed.ConclusionsIn summary, we reported on a simple, efficient method for preparing protoplasts of whole-organ tissue from tea plant. The findings are expected to contribute to the rapid development of tea plant biology.
Highlights
Plant protoplasts, totipotent, viable cells from which the cell walls have been enzymatically or mechanically removed, are targeted for the fusion of exogenous nucleic acids and cell organelles [1, 2]
It is acknowledged that the lack of effective and sustainable methods for preparing tea plant protoplasts have seriously limited the development of tea biology
Natural tea plantation seedlings For protoplast isolation and purification, unlignified branches, tender leaves, and mature leaves of Shuchanzao cultivar were selected (Fig. 1b); tender leaves were picked from tea plants belonging to the Huangshanbaicha, Zijuan, Huangkui, and Shuchanzao cultivars of C. sinensis var. sinensis grown on the campus plantation, from September to October 2020 (Fig. 1b)
Summary
The optimal conditions were 1.5% (w/v) cellulase and 0.4–0.6% (w/v) macerozyme in a solution containing 0.4 M mannitol, enzymatic hydrolysis over 10 h, and an iodixanol concentration of 65%. Differences in preparation, with regard to yield and viability in the tissues (roots, branches, and leaves), cultivars, and cultivation method, were observed
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