Study On Tissue Culture And Genetic Transformation Of 96-â…¡ Camptotheca Acuminata Containing CMO

Camptotheca acuminata Decaisne is native tree species in South China and well-known to camptothecin (CPT) in the body. It is fond of heat and light and well-inhabit sunny and moist bedrock and not drought-resistant. So expanding the range of planting has certain difficulty. In the molecule level to cultivate high-quality breed of C. acuminata is important significance with means of genetic engineering.At present, it is a most mature and extensive transformation system of Agrohacterium tumefaciens-mediated. In this paper, an effective tissue culture regeneration system was established, by which plantlets can be regeneration from either axillary bud way or callus way. The regeneration mechanism in vitro was revealed. Furthermore. CMO gene that was drought resistant gene was transferred into 96-â…¡ C. acuminata that was introduced from U.S.A. by Agrobacterium tumefaciens-mediated. Drought- resistance of 96-â…¡ C. acuminata was improved through expression of the purpose gene. The results of the paper filled in the gaps of tissue culture and genetic transformation of C. acuminata in China, and provided a new, effective way for 96-â…¡ C. acuminata breeding and propagation. The main contents and results as follows:1 , After sterilized shoots were obtained from axillary bud, a series of factors that influenced 96-â…¡ C. acuminata tissue culture were studied. The tissue culture system was developed from two regeneration ways, by which plantlets can be regeneration from either axillary bud way or callus way, with the callus inducing rate over 95%, the callus differentiation rate over 90%, rooting rate over 94% and transplanting rate over 95%.2, The plantlets of 96-â…¡ C. acuminata can adapt to environment quickly after transplanting. Comparing to seedlings, they had more advantages of grow rate, photosynthesis , content of CPT.3, Transgenic plants were obtained by Agrobacterium-mediated transfer system from 96-â…¡ C. acuminata leaf. To select the resistant shoots, the concentration of kanamycin was 20mg/L in callus inducing culture and 30mg/L in rooting culture. Explants were pre-cultured for 3 days before infection. The pre-cultured explants were incubated for 15min in the bacterial suspension which concentration (OD₆₀₀) was 0.5 or so. Co-cultivation period was 3 days. The target gene integrated into the genomic DNA of 96-â…¡ C. acuminata was detected and confirmed by PCR and Southern blotting analysis. Genetic transformation rate was 6.7%.