Noriyuki Morohoshi Abstract

We have tried to form a super-tree by new biotechnological and genetic engineering techniques. The first object of our research is to form a tree with less lignin content,by controlling the lignin biosynthesis genes using antisense RNA.

We have succeeded in isolating and sequencing the phenylalanine ammonia-lyase (PAL), O-methyltransferase (OMT) and peroxidase (PO) genes from hybrid aspen (Populus kitakamiensis), and also isolating the promoter regions of these genes. These results show that the genes of PAL, OMT and PO involved in lignification are palg2a, homt1 and prxA3a, respectively. We have been able to construct a system which includes transducing a foreign gene to the hybrid aspen by use of the Ti plasmid and infecting with Agrobacterium tumefaciens by the leaf desk method.

In this paper, we focus on the peroxidase gene. First, transgenic poplars were made using prxA1 of a peroxidase gene and the CaMV 35S promoter by the antisense RNA method. The transgenics could not grow to young plants, because the promoter cannot control its expression in situ and temporally. Therefore, a new vector having the original peroxidase (prxA3a) promoter and the antisense prxA3a gene, involved in lignification, was constructed. The transformants with this vector can grow as well as non-transformants.

The transgenic poplars have lower total peroxidase activity (10-25%) than that of the control. From the result of peroxidase isozyme analysis by isoelectric focusing, a peroxidase band (pi 3.8) involved in lignification disappeared in the transgenic plants. Lignin content in transgenic plants decreases 40-80% compared with control (100%), on the basis of potassium permanganate oxidation results. On the other hand, the amount of glucose determined by the alditol acetate method in transformants increases 5-10% compared with non-transformants.

These results show that it is possible to form transgenic poplars having lower lignin content and higher glucose content, which indicates the cellulose content.

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