Induction of hairy roots and characterization of peroxidase expression as a potential root growth marker in sesame.

Using hypocotyl and cotyledon of sesame seedlings, hairy root cultures were established and cDNA coding for a peroxidase was cloned from the roots. The frequency of sesame hairy root formation was higher in hypocotyl (33.4%) than cotyledon (9.3%). Applicable levels of kanamycin and hygromycin as a selectable marker were 100 microg/mL and 30 microg/mL, respectively. The peroxidase cDNA showed relatively high sequence identity with and similarity to plant class III peroxidase family. The cDNA encoded polypeptide was identified with the presence of three sequence features: 1) the putative 4 disulfide bridges, 2) an ER-targeted signal sequence in the N-terminus, and 3) two triplets, NXS for glycosylation. A real-time RT-PCR exhibited an abrupt increase in the peroxidase transcription activity after 4-week cultures of the sesame hairy roots and its highest level in 6-week cultured hairy roots. In contrast, the growth pattern of sesame hairy roots showed a typical sigmoidal curve. The active hairy root growth began after 2-week culture and their stationary growth phase occurred after 5-week culture. These results suggested that the peroxidase expression patterns at its transcription level could be used a potential indicator signaling a message that there will be no longer active growth in hairy root cultures. The sesame peroxidase gene was differentially expressed in different tissues.

Intrapericardial administration of adenovirus for gene transfer.

Gene transfer to the heart has been accomplished with intravascular administration of adenoviral vectors into the pericardial sac, by increasing the duration of exposure to the adenovirus, would result in gene expression in the pericardium and perhaps myocardium and therefore might provide an alternative method to intravascular administration for gene transfer. We injected a replication-deficient adenovirus (average 1 x 10(12) particles/ml in 3% sucrose; 1 x 10(10) plaque forming units/ml containing cDNA encoding a nuclear-targeted bacterial beta-galactosidase into the pericardial sac of dogs. Samples of the pericardium and heart were examined for enzymatic activity of beta-galactosidase and after histochemical staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. One day after injection of the adenovirus (1-3 ml), beta-galactosidase activity was highest in the parietal pericardium and left atrial tissue and lower in the right and left ventricles. Histochemical expression of the transgene was predominantly in the visceral pericardium of atria and ventricles and occasionally in the epicardial myocytes, arterioles, and venules. Pretreatment with doxycycline (5 mg) before adenovirus administration increased transgene activity in left ventricles. Thus adenovirus injected into the pericardial sac provides an effective method for gene transfer to the visceral and parietal pericardium over atria and ventricles.