Hevea Genetic Transformation System And Arabidopsis Thaliana CBF Gene Transfer By Agrobacterium Tumefaciens In Hevea

Rubber tree (Hevea brasiliensis) is an important economic crop in tropics. It has very important value especially in the fields of industry and military. At present, the consumption of natural rubber in China increases annually, and the shortage increases annually. Cold injury is the key factor that restricts the growth and distribution of rubber. Routine breeding of rubber tree can only partially, but not radically settle the problem. Gene engineering breeding becomes the core content of rubber tree biotechnological research and variety improvement.The technique of somatic embryogenesis in Hevea brasiliensis was succeeded in 1970s. But now the technique is dropped behind because that so far, only a few the clones can produce somatic embryogenesis through this technique, and the rate of the in vitro plant regeneration in most of the clones is very low. This becomes the main obstacle in the way to found the high genetic transformation system for reconstructing the inheritance of Hevea in molecule level. Compare of studies on model plants and economic crops, there are lags in advances in molecular biology of rubber tree.In this research item,we chosed the rubber variety of RY 7-33-97 widely generalized in Hainan, China. CBF gene, its downstream COR15a gene and regulating element CRT/DRE in promoter of COR15a gene, were transfered to firsthand calli from anther or inner integument culture and the long-term proliferated embryogenic calli via somatic embryogenesis by Agrobacterium tumefaciens to develop cold endurance rubber trees and to establish of genetic transformation system in Hevea including efficient culture via somatic embryogenesis. The results were shown as follow.1. The anther culture media of RY 7-33-97 was optimized.2. The long-term proliferation technique of Hevea embryonic calli was successfully established. The technique could increase the efficient of Hevea tissue culture, carve out a new way to found the high Hevea genetic transformation system and chronically afford plentiful good material for Hevea embryogenic cell suspension culture and protoplast culture.3. On the basis of Hevea anther culture and long-term proliferation technique of Hevea embryonic calli, we used the firsthand calli of Hevea anther and long-term proliferated embryonic calli to transfer by Agrobacterium tumefaciens. The result showed that embryonic calli were consumedly superior to the firsthand calli in transformation. And we have gained transformed embryoids via long-term proliferated embryonic calli. The study caved out a new way to found the high Hevea genetic transformation system.4. The study explored or optimized the index of every step of transformation mediated by Agrobacterium tumefaciens.5.This research laid a foundation for researching on fouction of CBF1,CBF3,ApCOR15a and provided a new method for cold endurance, presenting theorical significance and applicative prospect.