Gene expression levels of transgene 35S-gshI (γ-glutamylcysteine synthetase) cloned
from E. coli, and the endogenous gene gsh1 of poplar (Populus x canescens) were upregulated
by the DNA demethylating agent DHAC (5,6-dihydro-5'-azacytidine
hydrochloride) (10-4 M for 7 days) in aseptic leaf discs cultures. Two 35S-gshI-transgenic
(6lgl and 11ggs) and wild type (WT) poplar clones were used. The efficiency of gene
upregulation was also analyzed under herbicide paraquat stress (4 x 10-7 M). Levels of
gshI-mRNA and gsh1-mRNA were determined by RT-qPCR (reverse transcriptase
quantitative PCR) after cDNA synthesis. For internal control, the constitutively expressed
housekeeping poplar genes α-tubulin and actin were used, and the 2−HHCt method was
applied for data analysis. In long term DHAC treatment (21 days), a morphogenetic
response of de novo root development was observed on leaf discs in a wide concentration
range of DHAC (10-8 to 10-6 M). Adventitious shoots (11ggs clone) also emerged from
leaf discs after a combined treatment with DHAC (10-4 M) and paraquat (10-7 M). Shoots
were dissected, rooted and transplanted in glass houses for further analyses for
phytoremediation capacity. Since DNA methylation patterns are inherited (epigenetic
memory), these poplar plants with increased gene expression levels of both transgene
35S-gshI and endogenous gene gsh1 provide novel plant sources for in situ application
