Hormonal and morphological changes during seed development of Cypripedium japonicum

With the goal of evaluating the factors that may be influencing the reproductive success of an endangered, terrestrial orchid Cypripedium japonicum Thunb., the anatomical and morphological characteristics of seed development were assessed, as were the levels of endogenous hormones to investigate the possible relationship between hormones and seed development. The results showed that morphological characteristics of seeds were changing from milky white and filiform to brown and fusiform. The development of ovule that initiated on the fertile valves only began after pollination, and a mature embryo sac was produced at 40 days after pollination (DAP). Fertilization occurred 50 to 60 DAP, and the newly formed zygote divided into two unequal cells that became the embryo proper and the suspensor. Seeds reached maturity at about 115 DAP, at which point they consisted only of a globular embryo and two layers of seed coat, without endosperm. The suspensor was lost, and only lipid particles and a large number of starch grains distributed in the cytoplasm. The peak of gibberellic acid (GA₃), zeatin riboside (ZR), and indole acetic acid (IAA) occurred in a time sequence, as 20, 30, and 40 DAP. There was a highly significant positive correlation among them (P < 0.01). Abscisic acid (ABA) content increased at 60 DAP and reached the maximum at 115 DAP. ABA contents were significantly negatively correlated with the three hormones above (P < 0.05). It indicated that the lack of endosperm and suspensor tissues, delayed differentiation of embryos, and high levels of ABA may lead to the difficulty of germination.