The Preliminary Studies Of Tissue Culture And Genetic Transformation On P.lactiflora Pall

The genus paeonia is highly valuable as ornamental and medical spieces. However, the long vegetative cycle and low propagation is a hinderance to both domestic and international markets. Therefore we should set up a perfect tissue culture system to solve these problems.In this study, embryos and shoots were used as explants to induce callus and adventitious buds, and the histological and physiological characteristics were investigeated during adventitious buds formation. Besides, the ipt gene was introduced into the callus by the Agrobacterium-mediated method.1. The callus inductionEmbryo was used as explant to induce callus, and the effects of several components, including 6-BA, 2,4-D, NAA, caseinhydrolysat and sucrose, on callus induction were carefully investigated by orthogonal design. The results showed that, 6-BA played the most important role in callus induction, then followed by the caseinhydrolysat, NAA, sucrose and 2,4-D respectively. Thererfore, shoots were sucessfully gained from the induced callus after adding 0.1~2.0 mg·L-1 of TDZ in the differentiation medium.2. Adventitious buds inductionShoots were used as explants in this investigation with the fixed concentration of 0.5mg·L-1 for 6-BA, and effects of different concentrations of GA3, sucrose or glucose were examined by orthogonal rotation combination design. The results revealed that, glucose was the most important among these factors, while the effects of the others couldn't be neglected. The optimum combination of above factors was as follows, GA31.84mg·L-1, sucrose 10 g·L-1 and glucose 18.41 g·L-1. The highest adventitious buds inducing ratio was 3.03 in theory after 40 days of culture.3. Changes of the histology and physiology during adventitious buds inducingHistological and physiological characteristics changes during adventitious buds inducing were studied with shoots as explants. Results showed that the adventitious buds was origined through the pathway of direct organogenesis, and the soluble proteins were decreased while the POD and SOD were increased during the adventitious buds forming.4. The study of the genetic transformation on callus by Agrobacterium-mediated methodThe ipt (SAG1,2-IPT) gene was transformed into callus by Agrobacterium-mediated method. Among the 72 callus treated, 18 well developed and multiplied were screened by D4 medium with Basta. Seventeen of the 18 callus were preliminarily identified as transformed by PCR.