Abstract
A major barrier to plantlet regeneration using somatic embryogenesis technology in Pinus massoniana is the recalcitrance to achieve efficient proliferation and germination of embryogenic cells, resulting from the loss of embryogenic potential (capability to form viable somatic embryos) during successive proliferation culture. Here, an integrative analysis of molecular and physiological mechanisms on the loss of embryogenic potential following long-term proliferation culture was performed to identify key factors involved in influencing proliferation and germination of embryogenic cells. Embryogenic potential of cells, which was identified by proliferation coefficient (PC) and germination percentage (GP) at the present study, was distinct between the 10th (S10, PC: 11.0, GP: 66.4%) and 30th (S30, PC: 1.3, GP: 4.6%) proliferation cycle given the duration of proliferation cycle was 14 days. Transcriptome analysis indicated 99.6% differentially expressed genes in reactive oxygen species production pathway were upregulated. Following an application of 1.0 mM antioxidant against H2O2, L-glutathione, in the culture media, increases in both PC and GP were investigated for S30 embryogenic cells. Further evidence revealed that H2O2 regulated Ca2+ signal by increasing Ca2+ influx and decreasing Ca2+ efflux, resulting in cytosolic Ca2+ accumulation. With the reduction of Ca2+ concentration in media, both PC and GP of embryogenic cells were significantly enhanced. Our findings suggest that H2O2 and Ca2+ get involved in the loss of embryogenic potential during long-term proliferation culture. We have developed information demonstrating that regulation of Ca2+ level and oxidation environment in vitro can enhance proliferation and germination of embryogenic cells in P. massoniana.
Key message
H2O2 and Ca2+ got involved in the embryogenic potential loss of cells during successive proliferation culture in Pinus massoniana by comparing transcriptomes, H2O2 and Ca2+ flux, and Ca2+ subcellular localization.
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Data Availability
RNA-seq datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI Sequence Read Archive (SRA) platform (http://trace.ncbi.nlm.nih.gov/Traces/sra/) under the accession number PRJNA761875. In addition, other original contributions presented in the study are available from the corresponding author upon request.
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Acknowledgements
We thank the local Paiyangshan Forest Farm for their valuable fieldwork and collection of plant materials. This work was supported by the National Natural Science Foundation of China [32260381 and 31960311], Guangxi Science and Technology Project [2023GXNSFAA026449], and Guangxi Forestry Science and Technology Project [(2021)3].
Funding
This work was funded by the National Natural Science Foundation of China [32260381 and 31960311], Guangxi Science and Technology Project [2023GXNSFAA026449], and Guangxi Forestry Science and Technology Project [(2021)3].
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YW performed the data analysis and manuscript drafting. RY conceived the project, designed the research, and interpret the results. All authors have read and approved the manuscript.
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I certify that this manuscript is original and has not been published and will not be submitted elsewhere for publication while being considered by Plant Cell, Tissue and Organ Culture. And the study is not split up into several parts to increase the quantity of submissions and submitted to various journals or to one journal over time. No data have been fabricated or manipulated (including images) to support our conclusions. No data, text, or theories from others are presented as if they were our own. The submission has been received explicitly from all co-authors. And authors whose names appear on the submission have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for the results.
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Wang, Y., Yao, R. H2O2 and Ca2+ are involved in the embryogenic potential loss of cells during long-term proliferation culture in Pinus massoniana. Plant Cell Tiss Organ Cult 154, 657–672 (2023). https://doi.org/10.1007/s11240-023-02540-4
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DOI: https://doi.org/10.1007/s11240-023-02540-4