| Citrus medica L. var. sarcodactylis is a variation of Fructus Citri which belongs to Citrus of Rutaceae. It is not only a curiosa for its aroma and fruit, but also a rare Chinese traditional medicine. It is mainly cultivated in Guangdong, Fujian, Sichuan and Zhejiang province. The fruit produced in Jinhua, Zhejiang has the most excellent quality. But the plant can not bear the cold in winter. As a result, the majority of Citrus medica died, and the quality and quantity of the plant were influenced severely. With the rapid development of modern bio-technology, gene-project provide efficient methods and access for resolving the problem. Agrobacterium-mediated transformation and particle bombardment were studied in this study.　The factors which influence two transformation methods were discussed respectively, and optimized transformation conditions, We primarily established Agrobacterium-mediated transformation system , and　several main parameters impacting particle bombardment were confirmed. We detected foreign gene integrated into explant by PCR technology, In the mean time, we also detected the target gene that had been expressed in bergamot's callus accompanying with NPTII gene by GUS report gene. It provided the evidence that was used molecular biology for transformation. The main results were as follows: 1.Taking bergamot's leaves as explants, we selected the suited regeneration basic media that is MT, the best inducing callus hormone combination is 1.5 mg.L-12,4-D,1.0mg.L-1 NAA and 0.4 mg.L-16-BA. The　inducing frequency is 98.1 percent. the best differentiation hormone combination is 5 mg.L-16-BA and0.5 mg.L-1IAA. 2.Factors that influence percentage of agrobacterium-mediated transformation including time of pre-culture, time of infection, time of co-culture, the kind and concentration of antibiotic , day of delaying screening culture, different screening pressure were studied. A grobacterium-mediated transformation system is constructed. Its contents are as follows: leaves was pre-cultivated 3 days in darkness, leaf segments should be infected 20 minutes in Agrobacterium tumefaciens with OD600=0.6-0.8, co-cultivated 3 days with the addition of 400mg.L-1 L-cys and 100μmol.L-1AS, delayed to screen 2-4 days with the addition of 250mg.L-1 Cef, 250 mg.L-1Cb, 400 mg.L-1L-cys,AS100μmol.L-1 , and screened with 100 mg.L-1 Km for selecting resistant leaves until get resistant callus, and transferred to light conditions and sub-cultivated once or twice, reproduced after obtaining restantant shoots. 3. Particle bombardment transformation: taking tender leaves as receptor, leaves were bombarded with 1.0μm diameter gold. We obtained the much higher frequency of transient expression of GUS, under the bombardment distance of 6cm, the vacuum pressure of about 26 inches Hg and the Helium pressure of 1100psi,and 48 hours of delaying culture conditions. The activity of GUS transient expression is also much higher. We found that bombardment times do not influence GUS transient expression obviously. 4.We obtained that Km-resistant callus by agrobacterium-mediated transferring TPS gene into bergamot's leaves. We used GUS and PCR to detect the transforming callus, the result showed that TPS gene had been transformed to bergamot's genome. In the agrobacterium-mediated transformation, the frequency of GUS transient expression is 5.9 percent, two pieces Km-resistant callus were detected TPS gene in eight pieces Km-resistant callus by PCR. In particle bombardment transformation , the frequency of GUS transient expression is 17.8 percent, but TPS gene was not detected by PCR.
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