Abstract
The proposed progresses of this research project in the first year are 1) Transgenic trees of eucalyptus (Eucalyptus camaldulensis and E. grandis x E. urophylla) and Jatropha curcas L. will first be propagated by both tissue culture and cuttings. Well rooted transgenic plants will then be cultured and preserved in isolated net-room. PCR and Southern analysis were carried out to confirm the transgene insertion patterns of different transgenic clones. 2) Establishment of an isolated net-room for further transgenic tree bio-safety assessment. 3) A key gene involved in Taxoid biosynthesis pathway will be cloned and constructed onto expression vector for further Agrobacterium-mediated transformation experiments. 4) Efficiency of taxoids (mainly BC) production will be evaluated by different bioreactors and culture methods to collect valuable data for building up effective scale-up production system in the future. Progresses by the end of this first year are: 1) 50 individual of transgenic and wild eucalyptus clones were propagated and preserved in isolated net-room for further analysis, meanwhile, 5 transgenic J. curcas clones harboring GUS reporter gene were propagated by tissue culture, and the evaluation of transplanting survival rate will be carried on. 2) The isolated-net room is under construction and will be finished by the end of this year. 3) The BAPT gene was cloned from Taxus sumatrana by reverse-transcription method, and the gene sequence had been registered online. The BAPT gene was constructed onto expression vector driven by CaMV35S in antisense direction and then transferred into Agrobacterium tumefaciens A281 and A. rhizogenesis AT15834 for further experiments. 4) 2 replications of culture experiments were performed by using 1L and 2L disposable bioreactor separately. There were no differences of cell growth rate in 1L and 2L disposable bioreactor. 5) 2 times of semi-continuous culture experiments were performed by using both disposable bioreactor and stir type bioreactor, and the cell growth rate and BC production were evaluated. Current results showed that in semi-continuous culture tests the yield of BC is the highest when cells were cultured in 2L stir type bioreactor.