Response of Rudimentary Zygotic Embryos of Ilex crenata Thunb. to Plant Growth Regulators

Seed germination of Ilex crenata Thunb. usually takes 2-3 years, which significantly limited the breeding and selection of new cultivars. Rudimentary zygotic embryos of Ilex crenata Thunb. ‘Sky Pencil’ seedlings were removed from dark purple mature fruits and cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without auxins (2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), and Indole-3-butyric acid (IBA)) or cytokinins (benzyladenine (6-BA), kinetin(KT), zeatin (ZT), thidiazuron (TDZ), and isopentenyladenine (2-ip)). All cultured embryos were incubated in darkness at 27 ±2°C and data were taken after 6 weeks. Auxins could induce zygotic embryo to seedlings (both shoots and roots), shoots, or calli. The maximum percentage (100%) of embryos germination was achieved when the media supplemented with 2,4-D at 0.1 mg·L^-1. Media with higher concentrations of 2,4-D (0.5-2.0 mg·L^-1) or NAA (1.0-2.0 mg·L^-1) induced significantly more calli than that of other treatments. When 2,4-D concentration was at 0.5 mg·L^-1, the highest shoots induction percentage (70.4%) was yielded. Cytokinins could not induce embryo to calli alone. Lower concentrations of ZT (0.1-1.0 mg·L^-1), TDZ (0.1-1.0 mg·L^-1), or 2-ip (0.1-0.5 mg·L^-1) promoted embryo germination. However, media containing 0.5-2.0 mg·L^-1 6-BA, 0.5-2.0 mg·L^-1 KT, or 2.0 mg·L^-1 2-ip were totally ineffective in inducing embryo growth. The highest shoots induction percentage was produced under treatment of ZT at 2.0 mg·L^-1. Comparing traditional seed germination, zygotic embryos could be germinated for weeks. We recommend 1/4MS media with 2,4-D at 0.1 mg·L^-1 for embryo germination and 2,4-D at 0.5-2.0 mg·L^-1 for callus induction.