Low temperature and dark culture inhibited explant browning and promoted in vitro regeneration of Juglans mandshurica

Juglans mandshurica is an important component of Korean pine mixed and secondary forests in Northeast China and is also a precious timber and economic species. In this study, plant regeneration of J. mandshurica has been established using stems with bud and mature zygotic embryos as explants. The results showed that placing the stems with buds in low-temperature conditions (0 to 4°C) and in darkness for 5 d inhibited the browning of the explants and promoted bud sprouting; Na₂S₂O₃ also inhibited the browning of mature zygotic embryos significantly. The suitable basal media and hormones for bud sprouting and zygotic embryo germination were (1) WPM medium (woody plant medium) containing 2.0 mg·L⁻¹ 6-benzylaminopurine (6-BA) and 0.01 mg·L⁻¹ 3-indolebutyric acid (IBA) and (2) DKW medium (Driver and Kuniyuki medium) containing 2.5 mg·L⁻¹ 6-BA and 0.1 mg·L⁻¹ IBA. The suitable basal medium and hormone for rooting of shoots from buds was 1/2 Murashige and Skoog (MS) medium (the macro-elements were half-strength of MS medium) supplemented with 1.0 mg·L⁻¹ IBA, and the suitable basal medium and hormone for rooting of seedlings derived from zygotic embryo was 1/2 MS medium containing 1.0 mg·L⁻¹ IBA, and DKW medium with 2.5 mg·L⁻¹ 6-BA, 0.1 mg·L⁻¹ IBA, 0.2 mg·L⁻¹ gibberellin A3 (GA₃), and 0.2 g·L⁻¹ activated carbon (AC). About 95% of the rooted plantlets survived after acclimation and transplantation. These results provided a foundation for the scale propagation to meet the demands of forestry production of the species.