Morphological and endogenous phytohormone changes during long-term embryogenic cultures in Korean pine

Abstract Somatic embryogenesis (SE) is a biotechnological tool with high application potential for in vitro propagation and regeneration of conifers species. However, in SE of Korean pine, a dominant species of the natural mixed coniferous and broadleaf forests of the temperate forests in Northeast China, decreasing embryogenic potential was found in embryogenic callus (EC) with successive subcultures, especially when the cultures were maintained for more than 5-6 months, which is one of the most significant problems preventing commercial implementation of the SE technique. Aiming to investigate the morphological and the endogenous phytohormone changes associated with the decreasing embryogenic potential of Korean pine, here, we compared seven types of calli during long-term embryogenic culture. It was found that both the external morphological characteristics and internal cell structure were significantly different among the different types of calli. Specifically, similar to their zygotic counterparts of conifer trees, the calli with high embryogenic potential were characterized by a higher number of designated proembryogenic masses (PEMs) structure. In addition, surface of this types of callus was covered with a large amount of mucilaginous extracellular matrix (ECM), which could further differentiate into globular embryos containing the embryonal masses (EM) and embryonic tube (ET) structure.In contrast, in those low embryogenic potential calli, we observed disintegrated PEMs structure, a large amount of irregularly shaped cells, and degraded cells, which failed to form somatic embryos. The endogenous hormone results showed that high levels of ABA were beneficial for promoting early somatic embryo differentiation in Korean pine, but maintaining high embryogenic potential required moderate levels of IAA and low levels of ZR. Additionally, a high ratio of IAA / ABA and MeJA / ABA was conducive to maintaining high embryogenic potential, whereas a low ratio of IAA / ABA and MeJA / ABA were effective for early somatic embryos morphological differentiation. This study demonstrates morphological and physiological characteristics of the embryogenesis potential of Korean pine calli.