Establishment of PEG-Mediated Transient Gene Expression in Protoplasts Isolated from the Callus of Cunninghamia lanceolata

Abstract

Cunninghamia lanceolata (C. lanceolata) is an important timber tree species in southern China that requires gene function studies to understand its traits. In this study, we investigated the callus induction rates of immature zygotic embryos from reciprocal hybrids between genotypes B46 and B49. With zygotic embryo development, the callus induction rates showed an increasing trend, followed by a decreasing trend. Moreover, the rate of callus induction in genotype B46 × B49 immature zygotic embryos was greater than in genotype B49 × B46. Callus from C. lanceolata with genotype B46 × B49 was selected as the donor material for protoplast isolation. By using an enzymatic digestion solution containing cellulase, macerozyme, and pectinase, combined with an osmotic stabilizer, we obtained 9.76 × 106 protoplasts/mL with 92.7% viability. We subsequently transformed plasmids into C. lanceolata callus protoplasts and observed the location of the H2B-eYFP fusion protein in the nucleus. To achieve transient transfection of C. lanceolata callus protoplasts, we compared transfection efficiencies at different concentrations of PEG4000, PEG6000, or PEG8000 in a modified MMg solution. We found that 20% (w/v) PEG6000 mediated the transient transfection of C. lanceolata callus protoplasts with a 6.70% efficiency. This study provides a technical foundation for future research on transient transfection and functional analysis of C. lanceolata genes.