A sorbitol protection assay (Onishi et al., 2000) was done with three representative alkamides, alk3a, alk5, and alk11, and caspofungin and hygromycin
B to further test cell wall disruption activities.
In order to produce the second construct containing COL1A2, Hygromycin
resistant gene and also [pPICZ.sub.[alpha]]A([Amp.sup.R]) which didn't include zeocin, were both flanked by artificial cut sites, and then digested with NheI and AflII.
Total soluble protein extraction from fresh leaves, protein quantification, thin layer chromatography (TLC) assays for hygromycin
phosphotransferase (HPT) and phosphinothricin acetyltransferase (PAT) enzymes were performed following the procedures described by Datta et al.
A total of 11 independent T0 hygromycin
resistant transformed calli were successfully recovered from the selection media and regenerated as described previously (Fig.
tumefaciens was studied and the sensitivity to kanamycin is presented and compared with hygromycin
After selection in hygromycin
B-containing growth medium, four clones were selected for further characterization.
Putative gene deletion mutants were recovered and selected on a complete medium with 200 ug/mL hygromycin
and further selected by three rounds of screening using PCR (1st round), Southern blot (2nd round), and RT-PCR (3rd round).
It also contains the selectable marker genes hygromycin
phosphotransferase (hyg) and kanamycin (kanr).
When these inoculated seeds grew to three leaves, a 1 hygromycin
solution was used to brush the second and third leaf of inoculated seedlings to identify transformants.
pBIG3C  containing hygromycin
B resistant gene hph was kindly gifted by Professor Youliang Peng (China Agricultural University).
Selection and [beta]-D-Glucuronidase Screening