Abstract:

Via sequential single-gene transformation strategy, antisense phospholipase Dγ(Anti-PLDγ)gene and chitinase (CH5B) gene were introduced into Populus deltoides (G2) in proper order by Agrobacterium tumefacien mediated so as to enhance salt-tolerance and disease resistance. Firstly, the optimal media of P. deltoides (G2) for bud differentiation and rooting were established. The sensitivity of explants to kanamycin and hygromycin were tested separately in order to decide a reasonable selective pressure for transformation. Anti-PLDγ gene was introduced firstly. 21 kanamycin-resistant rooted plants were obtained through successive selection in shoot and root induction stage under high level kanamycin pressure. PCR and PCR-Southern analysis showed that 13 of 21 kanamycin-resistant rooted plants were positive. This meant that Anti-PLDγ gene was successfully integrated into the genome of these 13 plants. Salt-tolerance test demonstrated that 4/13 plants were enhanced to some extent. The No.4 plant was selected again to introduce chitinase (CH5B) gene. After successive selection in shoot and root induction stage under high level hygromycin pressure, 6 hygromycin_resistant rooted plants were gained. PCR and PCR-Southern analysis showed that 6 of these rooted plants were positive. This meant that CH5B gene was also successfully integrated into the genome of P. deltoides (G2). The CH5B gene and reporter gene GUS are in the same open reading frame (ORF), so via histochemical staining of GUS gene test, demonstrated that 6 plants can express CH5B gene normally.

Key words: Populus deltoides;antisense phospholipase D&gamma, gene(Anti-PLDγ);chitinase gene (CH5B);salt-tolerance;disease resistance