The role of the walnut (Juglans regia L.) shell in nut development, transportation, cleaning, and storage is often ignored. The shell suture seal and thickness are directly associated with kernel characteristics. In the present study, shell differentiation and microstructure were observed with an optical microscope using paraffin-sectioning and cryosectioning. The results showed that the parenchymal cells of the endocarp began to differentiate into sclerenchymal cells from 49 d after flowering (DAF), and the entire process continued until fruit maturation. The mature shell consists of three parts, including the sclereid layer (L1), sclerenchymal cell layer (L2), and shrunken cell layer (L3), from the outside to the inside. The shell thickness, suture seal grade, and mechanical strength were evaluated, as well as the lignin, cellulose, and phenolic compounds of the shell. Suture seal grade was positively correlated with lignin content, shell thickness, and L1 thickness and negatively correlated with shell cell diameter. Similarly, the mechanical strength of the shell was positively correlated with lignin content and L1 thickness. 'Qingxing' fruits were subjected to two treatments, namely, 30% shading and 70% shading, from 10 d after anthesis to maturity, with no shading used as control. After harvesting in September, nutshell sections showed thinner shells, with decreased contents of lignin and polyphenols, obtained under shaded conditions, and two of the three parts of the shell changed dramatically. The thinning of L1 and thickening of L3 eventually led to a thinner shell. The aim of this study was to evaluate the relationship among the shell structure, cellular components, and physical properties and provide a theoretical basis for cultivar breeding, rational planting density, and regulation of shell development.

Background 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. Results 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. Conclusion 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.