In Vitro Culture Of Camellia Nitidissima Chi

In this experiment, the relatively systematic research of organogenesis in vitro and somatic embryogenesis from the stem segments of adult trees was carried out in Camellia nitidissima, and then the changes of protein components during the development of somatic embryogenesis were analyzed by SDS-PAGE. The main results were as follows:1. The system of organogenesis in vitro from the stem segments of adult trees was established in Camellia nitidissima. The results showed that the effective method of surface sterilization for the explants was discontinuous sterilization; the modified solid WPM medium supplemented with 2.0 mg·L^-1 BA and 0.5 mg·L^-1 IAA was suitable for inducing germination of stem auxiliary buds, and the rate of germination was up to 57.1%; the modified solid WPM medium with 3.0 mg·L^-1 BA and 0.2 mg·L^-1 IAA was suitable for multiplication of shoot; and the multiplication of shoots rooted in the solid 1/2MS medium supplemented with 4 mg·L^-1 or 6 mg·L^-1 NAA.2. It was first time to induce somatic embryos from the stem segments of adult trees in Camellia nitidissima, and the protocols for efficient and repetitive somatic embryogenesis, maturation and conversion of somatic embryos were developed.Direct somatic embryogenesis occurred from the stem segments of adult trees in Camellia nitidissima . Somatic embryos were induced on the solid MS medium containing the combination of 2 mg·L^-1 BA and 0.5 mg·L^-1 NAA, and then secondary somatic embryogenesis emerged from the primary embryos.There were three approaches to maintain the system of repetitive somatic embryogenesis: ① Path Ⅰ: this approach could be used for development of the globular embryos and proliferation of the early cotyledonary embryos, but the times of subculture were limited. ②Path Ⅱ: this approach could be used for the proliferation of all developmental stages of somatic embryos except for the globularembryos; and the proliferation rate was high, but the somatic embryos were highly unsynchronized. ?Path III: this approach could be used for the long-term maintenance and proliferation of the big cotyledonary embryos. The secondary somatic embryos almost stayed at the stage of the big cotyledonary stage for a prolonged culturing period. The suitable mediums for realizing these three approaches were: the modified solid ER with 0.7 mg-L''2,4-D and 30 g-L"1 sucrose, the modified solid ER adding 0.1 mg-L'1 NAA, 3 mg-L'1 BA, 30 -L"1 sorbitol, and the solid MS supplemented with 40 g-L'1 sucrose and 20 g-L'1 sorbitol, respectively. These three approaches were related and formed a system of repetitive somatic embryogenesis, the secondary somatic embryos could be subcultrued for a prolonged period.The maturation stage of somatic embryos included two step procedures: firstly, the small cotyledonary embryos were cultured on the solid MS medium containing 40 g-L'1 sucrose and 20 g-L"1 sorbitol, at this stage, the small cotyledonary embryos could develop into the big cotyledonary embryos; at the second step, the big cotyledonary embryos could mature after culturing on the solid MS medium containing 60 g-L'1 sucrose.The matured somatic embryos could germinate into small plants with the higher frequency of conversion on the modified solid WPM medium supplemented with 2.0 mg-L'1 BA and 0.2 mg-L"1 NAA, and the plantlets generated from somatic embryos also grew better on this medium.3. It was first time to classify the different types of somatic embryos during somatic embryogenesis in Camellia nitidissima. According to the morphological characteristics, somatic embryos could be classified into 5 types: normal embryos, cotyledon-abnormal embryos, linked-embryos, multiple-buds embryos and vitrified embryos. Flower-shaped-multicotyledon embryos is one of the cotyledon-abnormal embryos, they could develop into a kind of cotyledon-like leaves, which could be converted into normal leaves under the appropriate condition. This phenomenon hadn't been reported at somatic embryogenesis in other plants.