Cloning and transfer of the 4-coumarate:CoA ligase (4CL1) gene in Eucalyptus camaldulensis (3/4)

This project is proposed to produce transgenic Eucalyptus camaldulensis with characteristics of low lignin and high cellulose contents. In the last two years, the 4CL1 gene was successfully cloned from xylem tissues of E. camaldulensis by using RT-PCR technique. After sequencing and recognizing the correction of cDNA sequences, the gene was ligated in an antisense orientation onto the plasmid pBI121 to instead of the GUS gene, in which the CaMV 35S promoter had also been replaced with a xylem specific promoter. This reconstructed plasmid was then transferred into a binary strain of Agrobacterium tumefaciens. Transformation of the antisense Euc4CL1 gene was accomplished by co-cultivating the leaf pieces of E. camaldulensis with the Agrobacterium strain. Putative transformed callus was induced and primarily verified by PCR. Southern hybridization was used to confirm the existence of the recombinant gene in transgenic shoots regenerated from the putative transformed callus. In this third year, transgenic shoots will be rooted to produce in vitro plantlets, which will be readily moved to an isolated greenhouse through acclimation and hardening processes. These transgenic plants will be harvested when they are 4 month old. Lignin and cellulose contents will be measured.