포플러의 休眠枝 不定芽 誘導法을 이용한 GUS 遺傳子의 器外 形質轉換

Insertion and expression of GUS gene through in vivo transformation using adventitious bud regeneration technique of dormant scion in populus

We have developed the in vivo transformation method for Populus species without applications of in vitro tissue culture techniques. To induce sufficient number of adventitious shoots from dormant scions which were harvested from stool beds, several factors were tested : the length of scion, three different diameters of scion, three different wounding treatments on scion, presence or absence of axillary buds, cutting orientation. The best condition for adventitious shoot production was the scion without axillary buds, 25㎝ in length, 10-15㎜ in diameter with crooked cuts. Average 130 shoots were regenerated from the cut surface of one-year-old scion. Shoots were regenerated from callus-like structure originally induced from cambium of cut surface. A binary vector plasmid, pBI121, was used containing 2 chimeric gene constructs : 1) Nopaline synthase(nos) promoter-NPTⅡ-nos terminator and 2) CaMV 35S promoter-β-glucuronidase(GUS)-nos terminator. The binary vector was transfered to Agrobacterium tumefaciens strain A281 and C58 by triparental mating. The scions with wounding treatment were co-cultured for 24 hours with Agrobacterium binary vector systems in shaking incubator. After co-cultivation, these scions were washed and cultured on waterbeds. Following 14 days of culture in growth chamber, the regenerants from the co-cultivated scions were tested GUS assay to confirm the expression GUS gene. About 30% of regenerants were confirmed as transgenic poplar by the GUS activity in stems and leaves.