Study On The Callus Induction And Genetic Transformation Of Dianthus Caryophyllus L.

Carnation is a perennial herb flower which has a high ornamental value and now is one of four major varieties of cut flowers in the world. Relatively short vase life affected its ornamental value. Traditional preservation methods with a higher cost can prolong the vase life,but cause more problems about the environmental pollution. The application of genetic engineering technology in carnation will extend the longevity of cut flower fundamentally. Two experiments have been conducted. First, preliminary study on the induction of callus and the plant regeneration with the flower buds as explants was performed. Second, the ACO gene with different T-DNA structures was transferred to spray carnation cultivars via Agrobacterium tumeficient. The experimental results are as follows:(1) The Induction of Callus and DifferentiationUsing two cultivars of potted spray carnation with red and pink flower as materials, the factors that affect the generation of callus, including different varieties, concentrations and types of hormone, concentrations of sucrose, the sizes of explants, are analyzed. The callus of carnation is induced and further plantlet is appeared. The results show that the difference concentrations of plant growth regulators and their combinations make the significant difference in the induction rate of callus. The highest induction rate of callus obtained on the medium of MS+2.0mg/L 2,4-D+0.2 mg/L 6-BA+9% sucrose+7.5g/L agar, up to 85.1%; The size of explants plays an important role on the induction of callus. When the diameter of the buds is about 1.0-1.5cm, the induction rate is highest; To a certain extent, sucrose concentration affects the differentiation of the callus and the optimal concentration is 3%.(2) Genetic TransformationThe regeneration system of five carnation varieties, such as’Anjing’,’Huaerzi’, ’Huoxing’,’Xiangxiang’and’Taisi’, is established. There is significant difference of the regeneration rates between cultivars, and’Taisi’owns the highest rate of regeneration, up to 59%, the regeneration rate of’Huoxing’is the lowest (36%). The Agrobacterium tumefaciens-mediated genetic transformation system of carnation was established followed the procedure, leaf explants are pre-cultured on shoot-inducing medium for 2 days, immersed in Agrobacterium suspension for about 8-12 min, and.cocultured on regeneration medium containing 100μmol/L acetosyringone (AS) for 3 days, then the transgenic shoots are selected on selection medium with 4 mg/L hygromycin (Hyg) and 400mg/L cefotaxime (Cef). The results show that the differentiation frequency of adventitious shoots exists significant difference between cultivars, the highest rate of differentiation is 8.4% obtained from’Taisi’, the second is 5.4% from’Huaerzi’, and no plantlet was produced from’Huoxing’; 23 of 115 plantlets (20%) was positive for PCR. In addition, the impact of ammonium nitrate on the conversion rate is explored, and the rate of genetic transformation of carnation is increased through increasing the amount of ammonium nitrate.