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Yujie Yang, Donglin Zhang, Zhihui Li, Xiaoling Jin and Jinying Dong

To shorten Ilex seed germination time and speed up breeding cycles, immature embryos of Ilex crenata ‘Sky Pencil’ seedlings were removed from fruits at their heart-shape stage and cultured in vitro on Murashige and Skoog (MS) medium or Woody Plant Medium (WPM) with 3% sucrose and 0.65% agar. Cultures were incubated at 27 °C for 2 weeks in darkness and subsequently moved to a growth chamber with 14-hour photoperiod (115 μmol⋅m−2⋅s–1). Embryos began to germinate 2–3 weeks after culture. The highest germination rate was 91.67% under 1/4 MS medium. Embryos cultured on MS medium also had high germination rates and produced the longest seedlings to 8.02 mm. Nodal segments with one axillary bud taken from embryo germination seedlings were cultured on MS medium with various concentrations of cytokinins and auxins for micropropagation. Zeatin (ZT; 4-hydroxy-3-methyl-trans-2-butenylaminopurine) increased the number of shoots and shoot lengths significantly more than 6-benzylaminopurine (6-BA). The recommended ZT concentration should be 2.28 µM. Rooting induction could be established on 1/4 MS medium with various concentrations of indole-3-butyric acid (IBA) or 1-naphthaleneacetic acid (NAA). IBA at 4.14 µM produced the best rooting percentage (91.67%) and good-root quality. All rooted plantlets were transplanted into a mixture of peatmoss and perlite (1:1 v/v) and acclimatized in a mist system. The average survival rate was 88.8%. The rapid embryo germination protocol for Ilex crenata could save Ilex breeders at least 2 years compared with traditional seed germination.