H_2O_2 and Ca^2+ are involved in the embryogenic potential loss of cells during long-term proliferation culture in Pinus massoniana
H_2O_2 and Ca^2+ are involved in the embryogenic potential loss of cells during long-term proliferation culture in Pinus massoniana
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DOI：
10.1007/s11240-023-02540-4
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发表时间：
2023-07
期刊：
影响因子：
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通讯作者：
Yin Wang;R. Yao
中科院分区：
文献类型：
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作者：
Yin Wang;R. Yao
马尾松体细胞胚胎发生技术再生植株的一个主要障碍是胚性细胞在连续增殖培养过程中丧失了胚性潜能（形成有活力的体细胞胚的能力），难以有效地增殖和萌发。在这里，一个综合分析的分子和生理机制的损失，胚胎发生的潜力，长期增殖培养进行，以确定参与影响增殖和萌发的胚性细胞的关键因素。本研究采用增殖系数（PC）和发芽率（GP）两个指标对细胞的胚胎发生能力进行了鉴定，结果表明，在第10个增殖周期（S10，PC：11.0，GP：66.4%）和第30个增殖周期（S30，PC：1.3，GP：4.6%）之间，细胞的胚胎发生能力有显著差异，即增殖周期为14 d。转录组分析表明，活性氧产生途径中99.6%的差异表达基因表达上调。在培养基中加入1.0 mM抗H2 O2抗氧化剂L-谷胱甘肽后，对S30胚胎发生细胞的PC和GP增加进行了研究。H2 O2通过增加Ca 2+内流和减少Ca 2+外流调节细胞内Ca 2+信号，导致细胞内Ca 2+积累。随着培养基中Ca ~（2+）浓度的降低，胚性细胞的PC和GP均显著增加。本研究结果表明，H2 O2和Ca 2+参与了长期增殖培养过程中胚性潜能的丧失。我们已经开发的信息表明，在体外钙水平和氧化环境的调节，可以提高胚性细胞的增殖和萌发在P。马森尼亚。
A major barrier to plantlet regeneration using somatic embryogenesis technology inPinus massonianais 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 H2O2regulated 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 H2O2and 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 inP. massoniana.