| Carnation (Dianthus caryophyllus L.) and lisianthus(Eustoma grandiflorum (Raf.) Shinn) are important cut flowers, which have beautiful flower shape and rich colors, and become the people's favorite. In recent years, the impact of pests on flower production is more serious than before. The following factors caused the agricultural pests outbreak:the rapid development of flower industry, the frequent export-import and domestic trade, the different level of cultivation, and the various climatic conditions. Nowadays, cultivating pest-resistant transgenic plants based on plant genetic engineering technology has become a prominent mean to increase yield and quality of flowers. The insecticidal crystal protein gene of Bacillus thuringiensis (Bt) has been extensively studied and used in plant insect-resistant genetic engineering. The proteins encoded by Bt genes can specifically kill a variety of pests, such as Lepidoptera, Coleoptera, Diptera, acaridan, parasitic nematodes and protozoan. Bt proteins is harmless to human, animal and environment. More than five hundred Bt genes were isolated from many subspecies of Bacillus thuringiensis. At the same time, a few of Bts were reconstructed in order to be grately expressed in plants.In this study, the carnations, lisianthus and tobacco were the receptor materials, and the synthetic Bt genes cry1C* and cry2A* together with bar gene (herbicide resistance gene) were transferred into above three plant materials by Agrobacterium-mediated genetic transformation method. Through herbicide selection, PCR of genomic DNA and RT-PCR, we confirmed that the genes has inserted to genome of transformants and can be transcribed normally. Totally, six cry2A* transgenic carnation plantlets were obtained, and four cry2A* transgenic eustoma plantlets were obtained. For cry2A* transgenic tobacco, eight plantlets were gained. Indoor bioassay test results showed that cry2A* transgenic carnation has well resistance to spider mite, aphid and herbicide.The major findings of present study are as following:1. By freeze-thaw method, two plant expression vectors pBar-Cry1C* and pBar-Cry2A* were transferred into Agrobacterium strain LBA4404 and EHA105. The T-DNA region of vector pBar-Cry1C* have cry1C* and bar, and that of pBar-Cry2A* have cry2A* and bar. 2. Through comparison, the medium of carnation leaf regeneration in vitro was confirmed, which was MS medium with 6-BA (1.21mg/L), NAA (0.35mg/L), sucrose (30g/L), and agar (6g/L). The medium of lisianthus leaf regeneration in vitro was MS medium with 6-BA (0.4mg/L), IBA (0.1mg/L), sucrose (30g/L), and agar (6g/L). The rates of leaf regeneration were 92% and 93% separately.3. For selection of transgenic carnation, lisianthus and tobacco, the optimal concentration of Basta was determined by Basta gradient test, which were 200μL/L, 300μL/L and 200μL/L separately. In this study, the selected Basta was an agricultural herbicide, which content of active components is 18.5% only. So the optimal concentration of Basta to three transgenic plants were 0.37mg/L,0.56 mg/L and 0.37mg/L separately.4. Optimized the Agrobacterium-mediated genetic transformation of carnations and lisianthus. The parameters involved in Agrobacterium concentration, co-culture time, pre-culture and pre-culture time, co-culture medium and acetosyringone concentration. Finally, the optimal parameter of carnation were concentration of Agrobacterium OD600 0.6, co-culture time 72h, pre-culture time 24h, co-culture medium filter paper with water, and added 50mg/L AS to activation medium and co-culture medium. And that of lisianthus were OD600 0.6,72h, no pre-culture, filter paper with water, and added 50mg/L AS to activation medium and co-culture medium. It laid a foundation for the Agrobacterium-mediated genetic transformation of Bt gene into carnations and lisianthus.5. Using Agrobacterium-mediated genetic transformation method, Bt gene cry1C* and cry2A* together with bar gene were introduced into carnations, lisianthus and tobacco. After herbicide selection and PCR of genomic DNA,46 cry2A* transgenic carnation plantlets; 12 cry2A* transgenic lisianthus plantlets and 8 cry2A* transgenic tobacco plantlets were obtained. At the transcription level, positive transgenic plants were screened by RT-PCR method. Finally, the number of plants that the genes can be transcribed normally, was 6 lines, 4 lines and 8 lines separately. Unfortunately, no cry1C* transgenic plant was obtained. Possibly because of the infection ability of the Agrobacterium strain we selected was low. And the cycle time of Agrobacterium-mediated genetic transformation was longer. So we didn't have enough time to do that again. 6. Through indoor test of herbicide and insect resistance, cry2A* transgenic carnation showed good double resistance to insect and herbicide. The transgenic carnation plantlets have a good resistant to Basta by tests of leaf dip in Basta solution and Basta gradient medium culture. They could live normally in 600μL/L (1.11mg/L) Basta medium. Pest-resistant tests showed that transgenic carnation have a significant resistant to red spider and aphids, while the control plants have a number of spots, and roll in the leaves because of the pests occur.
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