Tissue culture is an effective method for the rapid breeding of seedlings and improving production efficiency, but explant browning is a key limiting factor of walnut tissue culture. Specifically, the polymerization of PPO-derived quinones that cause explant browning of walnut is not well understood. This study investigated explants of ‘Zanmei’ walnut shoot apices cultured in agar (A) or vermiculite (V) media, and the survival percentage, changes in phenolic content, POD and PPO activity, and JrPPO expression in explants were studied to determine the role of PPO in the browning of walnut explants. The results showed that the V media greatly reduced the death rate of explants, and 89.9 and 38.7% of the explants cultured in V media and A media survived, respectively. Compared with that of explants at 0 h, the PPO of explants cultured in A was highly active throughout the culture, but activity in those cultured in V remained low. The phenolic level of explants cultured in A increased significantly at 72 h but subsequently declined, and the content in the explants cultured in V increased to a high level only at 144 h. The POD in explants cultured in V showed high activity that did not cause browning. Gene expression assays showed that the expression of JrPPO1 was downregulated in explants cultured in both A and V. However, the expression of JrPPO2 was upregulated in explants cultured in A throughout the culture and upregulated in V at 144 h. JrPPO expression analyses in different tissues showed that JrPPO1 was highly expressed in stems, young leaves, mature leaves, catkins, pistils, and hulls, and JrPPO2 was highly expressed in mature leaves and pistils. Moreover, browning assays showed that both explants in A and leaf tissue exhibited high JrPPO2 activity. The rapid increase in phenolic content caused the browning and death of explants. V media delayed the rapid accumulation of phenolic compounds in walnut explants in the short term, which significantly decreased explants mortality. The results suggest that JrPPO2 plays a key role in the oxidation of phenols in explants after branch injury.

中文翻译：

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JrPPOs 在核桃外植体褐变中的作用

组培是快速育苗、提高生产效率的有效方法，但外植体褐变是核桃组培的关键限制因素。具体而言，导致核桃外植体褐变的 PPO 衍生醌的聚合尚不清楚。本研究调查了在琼脂 (A) 或蛭石 (V) 培养基中培养的“赞美”核桃芽尖的外植体，并研究了外植体中的存活率、酚类含量、POD 和 PPO 活性以及 JrPPO 表达的变化，以确定其作用PPO 在核桃外植体褐变中的作用。结果表明，V培养基大大降低了外植体的死亡率，在V培养基和A培养基中培养的外植体存活率分别为89.9%和38.7%。与 0 h 时的外植体相比，在 A 中培养的外植体的 PPO 在整个培养过程中高度活跃，但在 V 中培养的外植体的活性仍然很低。在A培养的外植体酚类水平在72小时显着增加但随后下降，而在V培养的外植体中的含量仅在144小时增加到高水平。在 V 中培养的外植体中的 POD 显示出高活性，不会引起褐变。基因表达分析表明，在 A 和 V 中培养的外植体中 JrPPO1 的表达均下调。然而，在 A 中培养的外植体中 JrPPO2 的表达在整个培养过程中均上调，并在 144 小时时在 V 中上调。JrPPO1 在不同组织中的表达分析表明 JrPPO1 在茎、幼叶、成熟叶、柳絮、雌蕊和外壳中高表达，JrPPO2在成熟叶和雌蕊中高表达。此外，褐变试验表明 A 和叶组织中的外植体均表现出高 JrPPO2 活性。酚类含量的快速增加导致外植体褐变和死亡。V培养基在短期内延缓了核桃外植体中酚类化合物的快速积累，显着降低了外植体的死亡率。结果表明，JrPPO2 在分枝损伤后外植体中酚类的氧化中起关键作用。