| Chinese narcissus (Narcissus tazetta var. Chinensis) belongs to Amaryllidaceae, which is a kind of perennial herb and one of famous traditional flowers in China. Because of its freshness, elegance and attractive appearance, Chinese narcissus is an important seasonal flower both in winter and spring, which is enjoyed by people. However, there are some perennial problems like single species and flower colour in Chinese narcissus, which can not meet the market demand. Genetic engineering is an effective way for variety improvement of Chinese narcissus.In this study, Zhangzhou (Fujian, China) narcissus was used as material. On the basis of establishment of the efficient regeneration system using scapes as materials, a preliminary study of transformation was carried out, in which scapes were chosen as explants; and pigments in flowers of Zhangzhou narcissus were also studied in this article, Using high performance liquid chromatography analysis, pigment composition and content of petals and deputy coronal were analyzed. These results will be the foundation for the cultivation new varieties of daffodils with gentic engineering method. The main results are as follows:1. Preparation of scape explants: Scapes begin elongation after the completion of bud differentiation in the mid of late September. Length and maturity of the scape were different in December, January and February. Scapes in the bottom of the bulb, connecting to the tender, are much easier to induce shoot. The length of scape in December is about 1 cm, 1-1.5 cm in January, and about 1.5 cm in February. Scapes were cut into 1-2 mm thin slices as explants.2. Establishment of efficient regeneration system of scapes: Directly bud induction of scape slices in different periods were studied, and the efficient regeneration system was established. Comparison of three different periods: December, January and February; correspondingly the most suitable medium for bud induction were MS basal medium with 5 mg·L-1 BA and 0.3 mg·L-1 NAA; MS basal medium with 5 mg·L-1 BA and 0.3 mg·L-1 NAA or MS basal medium supplied with 3mg·L-1 BA and 0.5 mg·L-1 NAA; MS basal medium with 3mg·L-1 BA and 0.5 mg·L-1 NAA, respectively. The differentiation rate can reach over 90% and differentiation factor was more than 5. It was also found that during inoculation scape slices need to be placed on the top side up. Growth of adventitious buds was found growing in the MS medium with 0.5 mg. L-1 BA and 0.3 mg. L-1 NAA, buds grew into strong small bulbs.3. The preliminary study on genetic transformation: Agrobacterium Tumefaciens EHA105 with pCMBIA2300-35S-LLFY-OCS was used in the transformation on Narcissus tazetta var. Chinensis. The experiments of inhibitory concentration gradient results showed that a concentration of 300 mg·L-1 Carbenicillin in the medium was enough, meanwhile the concentration of cefotaxime was at 500 mg·L-1; Experiment results show that the antibiotic concentration gradient which can inhibit the growth of Agrobacterium tumefaciens was 30 mg·L-1 kanamycin. Factors affecting the transformation efficiency were compared. Scapes were pre-cultured in MS medium with 5 mg·L-1 BA, 0.5 mg·L-1 NAA and 3% sucrose for 6 d, infected in Agrobacterium suspension (OD600 = 0.4) for 30 min, co-cultured for 4 d, after that screening was performed by transferring the scapes into the culture medium MS with 5 mg·L-1 BA, 0.5 mg·L-1NAA, 30 mg·L-1 Kan, 300 mg·L-1 Carb and 3% sucrose, using this transformation procedure, more resistant buds can be obtained.4. Flower pigment composition analysis: The carotenoids pigments in white petals and yellow deputy coronal of Zhangzhou (Fujian, China) narcissus were extracted, its elements and contents were determined by high performance liquid chromatography. The extract solution was the mixture of Methanol, ethyl acetate and petroleum, with the ratio of 1: 1: 1. At low temperature the carotenoids can be separated very well. Chromatographic conditions: column Luna (C18) was selected as reversed phase column (5μm 4.6 * 250 mm), the mobile phase was 0-10 min; acetonitrile: methanol: dichloromethane = 70: 25: 5, 20-30min; gradient was acetonitrile: methanol: dichloromethane = 45: 10: 45. Detection wavelength: 445nm, flow rate: 1.0 ml / min, column temperature: 25℃. The results showed that the main components of the Chinese daffodil pigment are lutein andβ-carotene. In yellow deputy coronal the content of lutein is much higher than that ofβ-carotene, however, both lutein andβ-carotene in white flowers are little.
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