The thin cell layers (TCLs) of zygotic embryos of Aesculus hippocastanum were cultured on Murashige and Skoog (MS) medium with and without 2,4-D. Somatic embryos developed directly on the TCLs of zygotic embryos at the highest frequency on hormone-free medium. The frequency of direct somatic embryo formation was suppressed by 2,4-D treatment. Microscopic observation revealed that compact and nodular structures formed on epidermal surfaces of TCLs, and these structures further developed into somatic embryos without the intervention of the callus stage, indicating direct somatic embryogenesis. Somatic embryos were not formed on the internal excised portion of zygotic embryos. Similar to zygotic embryo culture, direct somatic embryogenesis occurred from the culture of TCLs excised from cotyledonary somatic embryos on hormone-free MS medium. A similar suppression of direct somatic embryo development was observed in the presence of 2,4-D. Instead, friable embryogenic callus formation from TCL culture of somatic embryos was stimulated by 2,4-D treatment. Well-developed somatic embryos formed from the culture of embryogenic calluses. Dormancy of somatic embryos was observed because somatic embryos were not germinated on hormone-free medium without special treatment. Both cold treatment and GA₃ treatment resulted in the germination of somatic embryos.

在含有和不含 2,4-D 的 Murashige 和 Skoog (MS) 培养基上培养七叶树合子胚胎的薄细胞层 (TCL) 。在无激素培养基上，体细胞胚胎直接在合子胚胎的 TCL 上发育，频率最高。2,4-D 处理抑制了直接体细胞胚胎形成的频率。显微镜观察发现，TCL表皮表面形成致密的结节状结构，并且这些结构在没有愈伤组织阶段的干预下进一步发育为体细胞胚胎，表明直接体细胞胚胎发生。合子胚的内部切除部分未形成体细胞胚。与合子胚胎培养类似，直接体细胞胚胎发生是在无激素 MS 培养基上培养从子叶体细胞胚胎切除的 TCL 中发生的。在 2,4-D 存在的情况下，观察到对直接体细胞胚胎发育的类似抑制。相反，2,4-D 处理刺激了体细胞胚胎 TCL 培养物中易碎胚性愈伤组织的形成。发育良好的体细胞胚胎由胚性愈伤组织培养物形成。观察到体细胞胚的休眠是因为体细胞胚在没有特殊处理的无激素培养基上不会萌发。冷处理和GA ₃处理均导致体细胞胚的萌发。