This study cloned the PkSERK genes from Korean pine (Pinus koraiensis) and analyzed their expression characteristics, laying a foundation for functional research on PkSERKs in somatic embryogenesis of Korean pine.

The full-length coding sequence (CDS) of PkSERK1-3 gene in Korean pine was successfully cloned, subsequent bioinformatics analyses were conducted to examine the protein structure, physicochemical properties, and evolutionary relationships, subcellular localization was observed with green fluorescent protein (GFP) technology, additionally, and the expression patterns of these three genes in seven types of korean pine calli were analyzed by qRT-PCR technology.

PkSERK1, PkSERK2, and PkSERK3 were successfully cloned, with cDNA lengths of 1 824, 1 887 and 1 437 bp, encoding 607, 628 and 448 amino acids, respectively. PkSERK1 is an acidic, hydrophilic, and stable protein, while PkSERK2 is an acidic, hydrophilic, and unstable protein, and PkSERK3 is a basic, hydrophilic, and unstable protein; both PkSERK1 and PkSERK2 contain a signal peptide, whereas PkSERK3 does not have a signal peptide; these three proteins are all transmembrane proteins. The PkERK1 protein has the closest genetic relationship with ClSERK1-3 of Cunninghamia lanceolata, PkSERK2 has the closest genetic relationship with CsSERK1 of Camellia sinensis, and PkSERK3 has the closest genetic relationship with CjSERK3 of Cryptomeria japonica. PkSERK1 and PkSERK2 were located in the cell membrane, while PkSERK3 was located in the cell membrane and nucleus. The PkSERK1 gene was highly expressed in both callus with high embryogenic potential and callus with globular embryos of korean pine; PkSERK2 was highly expressed in brown callus; PkSERK3 was only highly expressed in callus with high embryogenic potential.

The genes of PkSERK1, PkSERK2 and PkSERK3 were cloned from korean pine, PkSERK1 may regulate the maintenance of embryogenic potential in callus and early globular embryo differentiation in Korean pine, high expression of PkSERK2 may be associated with browning embryogenic callus (EC) and early globular embryo differentiation in Korean pine, while PkSERK3 may only play a role in the maintenance of embryogenic potential in Korean pine callus.

We investigated the physiological characteristics of non embryogenization and somatic embryogenesis (SE) differentiation initiation in long-term subcultured embryogenic callus (EC) of Pinus koraiensis, explored the correspondence between morphological and physiological characteristics of the different embryogenic potential calli, which can provide a reference for overcoming the technical bottleneck that the embryogenetic potential of EC is difficult to maintain in SE of P. koraiensis.

In this study, different types of calli (white calli subcultured for 2 months and 6 months, yellow calli, water-stained calli, fibrotic calli, browned calli, and calli containing early spherical embryos, respectively) obtained by long-term subgeneration of . koraiensis were used as materials, and the differences in morphological characteristics, physiological indexes, and somatic embryo differentiation ability of different types of calli were compared and analyzed.

The six types calli of Korean pine obtained by subcultured for 2-6 months had significant differences in external morphological and histocytological characteristics such as color and texture, as well as proliferation rate and somatic embryo differentiation ability. In the 2-month-old white calli with high embryogenic potential (C1), a distinct proembryogenic masses (PEMs) structure was observed and globular embryos could be differentiated from the C1-type calli, whereas in the other five types of calli (C2-C6), the disintegration of PEMs structure, a large number of irregularly shaped cells and degraded cells were observed, there were no differentiation of globular embryos from the these 5 types of calli. The results showed that the contents of soluble sugar and starch were the highest in the calli with high embryonic potential, high activity of POD appeared in highly embryonic potential calli and browned calli, the activity of superoxide SOD was lower in the calli with high embryonic potential and the calli with differentiated globular embryos, while the activity of SOD was higher in the yellow calli with fast proliferation rate, the calli with differentiated globular embryos and the white calli with low embryonic potential had the highest CAT activity, the activities of SOD, POD and CAT in water-stained calli and fibrotic calli were low.

The long-term subculture of P. koraiensis EC to maintain embryonic potential or lose embryonic potential and the initiation of somatic embryo differentiation are synergistically regulated by intracellular protective enzymes and storage substances, and the high content of soluble sugar and starch are conducive to the maintenance of embryonic potential of P. koraiensis EC, and the roles of the three key enzymes SOD, POD and CAT in the formation of calli of P. koraiensis with different colors and textures are different.