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

Abstract

In SE of Korean pine, decreasing embryogenic potential was observed in embryogenic callus (EC) with successive subcultures, especially when the cultures were maintained for more than 5–6 months. Thus, aiming to investigate the changes in morphology and the endogenous phytohormone associated with the decreasing somatic embryo differentiation capacity of Korean pine, here, we compared Korean pine calli with different embryogenic potential. 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 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 abscisic acid (ABA) were beneficial for promoting early somatic embryo differentiation in Korean pine, but maintaining high embryogenic potential required moderate levels of indole-3-acetic acid (IAA), and low levels of zeatin-riboside (ZR). Additionally, a high ratio of IAA/ABA, methyl jasmonate (MeJA)/ABA, and gibberellins (GA3)/ABA were conducive to maintaining high embryogenic potential, whereas a low ratio of IAA/ABA and MeJA/ABA were effective for early somatic embryos morphological differentiation. The results can provide morphological and physiological indicators for identifying high embryogenic potential calli suitable for SE and help determine the best protocol on addition of exogenous hormones in the construction of a maintainable system of EC in Korean pine.