Studies On Establishment Of Efficient Regeneration System And Agrobacterium Tumefaciens-mediated Transformation Of Camptotheca Acuminata

Camptotheca acuminata of the family Nyssaceae is a native South China perennial deciduous tree,contain camptothecin(CPT) which possesses strong anti-cancer bioactivity. In this study , an efficient plant regeneration protocol in vitro and transformation with ipt gene mediated by Agrobacterium tumefaciens method of Camptotheca acuminata were achieved .The establishment of efficient regeneration system and Agrobacterium tumefaciens-mediated transformation of Camptotheca acuminata will make substantial contribution as an essential and useful means not only to further fundamental study of breeding by the application of genetic engineering technology but also to the establishment of genetically modified plants feasibly producing CPT.1. Establishment of high efficient regeneration system of Camptotheca acuminateIn this study.the explants of 10 days hypocotyls and 20 days cotyledon from aseptic seedings of Camptotheca acuminata were used to callus induce and shoots regeneration on woody plant medium(WPM). the most effective medium for callus induce and shoots regeneration was the WPM medium containing 1.5mg/L6-BA and 0.5mg/LNAA firstly, and then transferred to the medium with 1.0 mg/L 6-BA and 0.2 mg/L NAA.Callus Inductivity and differentiation maximum frequency were 99% and 77.8%.The shoot buds were multiplicated and elongated on medium with 0.5mg/L 6-BA, the rate of multiplication can reach 11.2 times. We found that medium containing 0.1-0.2mg/L 2,4-D could improve callus's growthing, but restrain the differentiation.and medium with 0.1-0.2mg/LKT was disadvantage for the shoots multiplication,elongation and rooting.the optimised rooting were obtained on medium with 0.5mg/LIBA, rooting rate was 98%.The best matrix for transplanting plants is one half field Soil pub half perlite rock,the survival rate of transplanting can reach 83.3%.2. Establishment the transformation System of Camptotheca acuminata Mediated by Agrobacterium tumefaciensThe genetic transformation system of Camptotheca acuminata was optimized by using the cotyledon and hypocotyl as receptors mediated with Agrobacterium tumefaciens.The content of kanamycin (Kan) selection pressures were determined as 30mg/L for shoot regeneration of cotyledon ,20mg/L for hypocotyls and 30mg/L for rooting.Timentin is superior to cefotaxime(cef) for shoot differentiation and A.tumefaciens elimination. The shoot formation rate were 42.8 % and 17.1% respectively when 300mg/L Timentin and cefotaxime were present in the regeneration medium.The suitable content of Timentin is the medium with 150 mg/L.To monitor the induction rate of Kanamycin-resistant callus, the optimal parameters (such as preculture time, bacterium concentration,infection time.co-culture time.)for A.tumefaciens-mediated transformation of C.acuminata were examined. The results showed that the best technical parameter was 5-20 days aged cotyledon and hypocotyls using for infection explants, 2d preculture time, 0.5(OD₆₀₀)bacterium concentration, 8-15min infection time and 3d co-culture time, the results showed that the extra gene ipt was integrated into C.acuminata genomes successful confirmed by PCR.