Key Laboratory of South China Agricultural Plant Molecular Analysis and Gene Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Int J Mol Sci. 2020 Dec 23;22(1):84. doi: 10.3390/ijms22010084.
In this paper, the development of the Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner integument cells were absorbed by the developing embryo. The Maudiae embryo sac exhibited an type of development. The time taken for the embryo to develop to a mature sac was 45-50 days after pollination (DAP) and most mature embryo sacs had completed fertilization and formed zygotes by about 50-54 DAP. transformation was achieved by injection of the ovaries by , resulting in 38 protocorms or seedlings after several rounds of hygromycin selection, corresponding to 2, 7, 5, 1, 3, 4, 9, and 7 plantlets from -mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, respectively. Transformation efficiency was highest at 50 DAP (2.54%), followed by 2.48% at 53 DAP and 2.45% at 48 DAP. Four randomly selected hygromycin-resistant plants were GUS-positive after PCR analysis. Semi-quantitative PCR and quantitative real-time PCR analysis revealed the expression of the gene in the leaves of eight hygromycin-resistant seedlings following -mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, while expression was not detected in the control. The best time to inject Maudiae ovaries with is 48-53 DAP, which corresponds to the period of fertilization. This protocol represents the first genetic transformation protocol for any species and will allow for expanded molecular breeding programs to introduce useful and interesting genes that can expand its ornamental and horticulturally important characteristics.
通过共聚焦激光扫描显微镜评估了授粉后不同发育阶段麻叶乌头胚囊的发育情况。麻叶乌头成熟种子由外种皮和球形胚组成，但没有胚乳，而内种皮细胞被发育中的胚吸收。麻叶乌头胚囊表现为 型发育。从授粉到胚胎发育成熟需要 45-50 天（DAP），大多数成熟胚囊在大约 50-54 DAP 时完成受精并形成受精卵。通过 注射到卵巢中实现了 转化，经过几轮潮霉素选择后得到 38 个原球茎或幼苗，对应于 30、35、42、43、45、48、50 和 53 DAP 时 -介导的卵巢注射的 2、7、5、1、3、4、9 和 7 株植物，分别。50 DAP 时转化效率最高（2.54%），其次是 53 DAP 时的 2.48%和 48 DAP 时的 2.45%。4 个随机选择的潮霉素抗性植物在 PCR 分析后均为 GUS 阳性。半定量 PCR 和定量实时 PCR 分析表明，在 30、35、42、43、45、48、50 和 53 DAP 时，-介导的卵巢注射后，8 株潮霉素抗性幼苗的叶片中表达了 基因，而对照中未检测到 表达。用 注射麻叶乌头卵巢的最佳时间是 48-53 DAP，这对应于受精期。该方案代表了任何 物种的第一个遗传转化方案，将允许扩大分子育种计划，引入有用和有趣的基因，以扩大其观赏和园艺重要特性。
