Study On The Agrobacterium-Mediated Cyclamen Transformation Of Chitinase Gene

Cyclamen is a world-famous flower, which is well popular with its abundant germplasm resource and various cultivars. For a long time, people do much continuous work in cyclamen breeding. Resistant breeding of cyclamen is a new direction. Fungous diseases do great harm to cyclamen and have serious influence on economic benefits.In the experiment young leaves were used as explants and Chitinase gene was delivered into cyclamen plants mediated by agrobacterium tumefaciens by means of leaf disk transformation. Regeneration and transformation system were established.Positive transgenic plants were obtained by PCR detection. The results were summarized as follows:1. Impacts on regeneration of Different hormone: 2,4-D and NAA, which belong to two kinds of auxins. Both 2,4-D and NAA can induce callus ,but the effect of 2,4-D is better than NAA, it is suitable for cyclamen.2. The optimum culture medium of establish regeneration: Callus induced medium is MS add 2,4-D0.5mg/L add BA0.2mg/L. Differentiation medium is MS add BA2.0mg/L add 2,4-D1.0mg/L add KT 0.2mg/L.Rooting medium is 1/2MS add NAA0.5mg/L.3. Determine selective pressure: The vector use NPTâ…¡gene as selectable maker gene ,it make transgenic plants against Kanamycin. Selective pressure of Kanamycin was 40mg/L in the experiment.4. The suitable condition of cyclamen genetic transformation system: After surface sterilization, leaf explants were precultured on induce callus medium MS add 2,4-D1.0mg/L add BA0.2mg/L for 3 days. Activated bacterial concentration is OD600=0.5 and infection time is 10 minutes. Co-cultivation medium is MS add acetosyringone 100uol/L and Co-cultivation time is three days. On the above-mentioned conditions can obtain relatively higher transformation ratio.5. Detection of transgenic plants: The primer was designed and synthesized according to the sequence of Chitinase gene. 4 positive plant between 6 regeneration plants by PCR detection. Positive rate is 66.7 percent, Its amounts 0.29 percent of total transformation explants. Positive rate is low. The result of PCR detection showed that Chitinase gene was integrated into cyclamen genome.